Gene encoding transglutaminase derived from fish

ABSTRACT

The present invention relates to a DNA fragment having a gene derived from fish which codes for a polypeptide possessing transglutaminase activity, a recombinant plasmid comprising a fish-derived DNA fragment which codes for a transglutaminase, a transformant into which a recombinant plasmid comprising a fish-derived DNA fragment which codes for a transglutaminase is introduced, and a method for the production of a transglutaminase, comprising culturing a transformant containing a fish-derived DNA fragment which codes for a transglutaminase.

This application is a continuation of application Ser. No. 08/004,729,filed on Jan. 14, 1993, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a DNA fragment having a gene derivedfrom fish which codes for a polypeptide possessing transglutaminaseactivity, a recombinant plasmid comprising a fish-derived DNA fragmentwhich codes for a transglutaminase, a transformant into which arecombinant plasmid comprising a fish-derived DNA fragment which codesfor a transglutaminase is introduced, and a method for the production ofa transglutaminase, comprising culturing a transformant containing afish-derived DNA fragment which codes for a transglutaminase.

2. Discussion of the Background

Transglutaminase (hereafter abbreviated as "TGase") is an enzyme whichcatalyzes an acyl group transfer reaction of a gamma-carboxyamido groupof a glutamine residue in a peptide chain. In the presence oftransglutaminase, an epsilon-amino group of a lysine residue in aprotein functions as an acyl receptor, thus formingepsilon-(gamma-Gln)-Lys crosslinkings in the protein, or if the lysineand glutamine residues are in two or more protein molecules,epsilon-(gamma-Gln)-Lys bridges are formed between the proteins.Alternatively, if a primary amine such as an amino acid, amino acidderivative, etc. functions as an acyl receptor, transglutaminaseintroduces the primary amine into the protein. Also, when waterfunctions as an acyl receptor, transglutaminase enzyme catalyzes thedeamidation or hydrolysis of a glutamine residue to a glutamic acidresidue.

TGase is used in the production of gelatinous food products andcosmetics, as well as yogurt, jelly and cheese, etc. (Japanese PatentPublication 50382/1989). Further, it is used for the production ofmaterials for thermostable microcapsules, carriers for immobilizedenzymes, etc.

A calcium (Ca²⁺)-independent TGase from bacteria of the genusStreptoverticillium has been discovered. Some concrete examples ofbacteria belonging to this genus include Streptoverticilliumgriseocarneum IFO 12776, Streptoverticillium cinnamoneum sub sp.cinnamoneum IFO 12852, Streptoverticillium mobaraense IFO 13819, etc.(Japanese Patent Application Publication 27471/1989).

Further, TGases derived from certain mammalian animals are also known.These include a TGase derived from guinea pig liver (Connellan, et al.,Journal of Biological Chemistry, Vol. 246, p. 1093-1098, 1971), fromhuman or bovine vascular endothelial cells (Gentile, et al., Journal ofBiological Chemistry, Vol. 266, p. 478-483, 1991, and Nakanishi, et al.,European Journal of Biochemistry, Vol. 202, p. 15-21, 1991), and fromhuman blood coagulation factor XIII (Takahashi, et al., Proc. Natl.Acad. Sci. USA, Vol. 83, p. 8019-8023, 1986).

Heretofore, the sources of TGase available for industrial use have beenmammals and bacteria. However, the products with which TGase processingare most common include processed marine (fish) products, a typicalexample of which is a boiled fish paste known as "kamaboko" (Seki, etal., Nippon Suisan Gakkaishi, Vol. 56, p. 125-132, 1990). The TGasebelieved to be responsible for the properties of "kamaboko" isapparently an enzyme derived from fish, present in the raw fishmaterials used in "kamaboko" preparation.

Prior to the present invention, no information has been availableconcerning a TGase gene from fish. However, a TGase from fish will bothwiden the range of TGase use and provide enzyme-processed fish productshaving similarities to natural fish products. Thus, the cloning,identification and expression of a fish TGase gene is highly desired,and may lead to a supply of fish-derived TGase at a low price.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide a novelDNA fragment which codes for a polypeptide derived from fish whichpossesses TGase activity.

A further object of the present invention is to provide a recombinantplasmid comprising a DNA fragment which codes for a polypeptide derivedfrom fish which possesses TGase activity.

A further object of the present invention is to provide a transformantcontaining a DNA fragment which codes for a polypeptide derived fromfish which possesses TGase activity.

A further object of the present invention is to provide a method for theproduction of a polypeptide derived from fish which possesses TGaseactivity, comprising culturing a transformant containing a DNA fragmentwhich codes for a polypeptide derived from fish which possesses TGaseactivity.

A further object of the present invention is to provide a purified andisolated polypeptide derived from fish which possesses TGase activity.

These and other objects of the invention which will become apparent fromthe following detailed description of the invention, have been achievedby a DNA fragment from fish which contains a gene which codes for TGase,expression of a gene which codes for TGase by genetic engineeringmethods, and culturing transformants transformed with an expressionvector containing a gene which codes for TGase.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 shows a restriction enzyme map for plasmid pSLTG5 which possessesthe acquired cDNA with a gene coding for Pagrus major transglutaminase;

FIG. 2 shows the correlation among cDNA clones which codes for Theragrachalcogramma (Alaska pollack) transglutaminase, and the restrictionenzyme map for the cDNA;

FIG. 3 shows a process for the construction of plasmid pIL6TG1, used forthe expression of Pagrus major transglutaminase;

FIG. 4 shows a base sequence listing of chemically synthesized DNA-1(SEQ ID NO:1). This shows the DNA base sequence which contains aconsensus SD (Shine-Dalgarno) sequence from E. coli and codes for theregion from methionine at the amino terminus of Pagrus majortransglutaminase to the 32nd amino acid, leucine;

FIG. 5 shows a process for the construction of plasmid pFTGN6, used forthe later construction of plasmid pTTG2-22, used in turn for theexpression of Pagrus major transglutaminase;

FIG. 6 shows a process for the acquisition of DNA fragments A, B and C,used in the construction of plasmid pTTG2-22, later used for theexpression of Pagrus major transglutaminase;

FIG. 7 shows a process for the construction of plasmid pTTG1;

FIG. 8 shows a process for the construction of expression plasmidpTTG2-22, used for the expression of Pagrus major transglutaminase;

FIG. 9 shows a process for the construction of plasmid pYSTG1, used forthe expression of Pagrus major transglutaminase cDNA in yeast;

FIG. 10 shows the restriction enzyme cutting sites in a cDNA clonecoding for transglutaminase derived from Theragra chalcogramma muscle;

FIG. 11 shows the restriction enzyme cutting sites in a cDNA clonederived from Paralichthys olivaceus liver;

FIG. 12 shows a plasmid carrying a cDNA fragment coding fortransglutaminase derived from Paralichthys olivaceus;

FIG. 13 shows a comparison between the thermal stability of Alaskapollack-derived transglutaminase (FTG) and that of guinea pig-derivedtransglutaminase (MTG), where ∘--∘ indicates the relative residualactivity of FTG, and -- indicates the relative residual activity ofMTG; and

FIG. 14 shows a comparison between the reactivity of Alaskapollack-derived transglutaminase (FTG) and guinea pig-derivedtransglutaminase (MTG) in polymerization of myosin H chain, where Δ--Δindicates the reactivity of FTG, □--□ indicates that of MTG, and ∘--∘indicates that of a control (no enzyme).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One recombinant transglutaminase for reforming food proteins is derivedfrom a guinea pig. This is a comparative TGase. Transglutaminasesderived from fish are superior to guinea pig TGase for industrial usesfor the following reasons:

(1) Fish has been eaten by human beings for a long period of time, evenraw. Therefore, the safety of fish transglutaminase is extremely high.Future costs for investigation of the safety of fish-drivedtransglutaminase may be smaller than that of one derived from otherliving things.

(2) Regarding the difference in the enzymatic characteristics betweenthe fish-derived transglutaminase and one derived from other mammals,especially for application thereof to fish paste products, specificallymentioned are the difference in the reactivity of the enzyme and theproduction cost to be influenced by the difference in the deactivatingconditions of the enzyme to be reacted (this will be referred to in thefollowing examples) and also the difference in the qualitative effectssuch as the natural taste and tooth feeling (expression of elasticity)of the reaction products. In fact, it has been found that a fishtransglutaminase has a high reactivity to a fish actomyosin (refer tothe following examples).

(3) The difference in the productivity of a recombinant transglutaminasewith microorganisms is also an important problem for consideration ofthe practical use of the transglutaminase. Ikura et al have alreadyreported the production of a guinea pig-derived transglutaminase withEscherichia coli (Eur. J. Biochem., Vol. 187, 705-711, 1990). However,the amount of it to be produced is extremely small. That is, thetransglutaminase in an extract of transglutaminase-producing cells wassuch that could only be detected with an antibody thereto (the amount ofthe transglutaminase to be produced is about 2.6 mg or so per liter ofthe culture medium). As opposed to this, where the DNA fragment codingfor the fish-derived transglutaminase of the present invention isapplied to a host of E. coli, it shows a productivity of producing about10% to 15% of E. coli-derived SDS-soluble total proteins, reaching anexpression quantity about 100 times or higher than that of the guineapig-derived transglutaminase. Thus, there is a high possibility that thefish-derived transglutaminase has a gene structure suitable toproduction of a recombinant one. (This will be explained in thefollowing examples.)

The above-mentioned various characteristics of the fish-derivedtransglutaminase yield great advantages in industrial use, especiallyfor reformation of food proteins therewith. The current shortage ofmarine resources, due to the 200 nautical sea mile restriction and thetotal fish catch quota restriction in the fishery sea area, causes aserious problem in the elevation of the raw material cost in producingmarine products. For instance, use of the enzyme of the presentinvention with other edible protein can reduce the concentration of thefish paste material in processed marine products, and lead to theeffective use of the underutilized marine resources.

On the other hand, the fish-derived transglutaminase is quite differentfrom a microorganism- or bacteria-derived transglutaminase (hereinafterreferred to as BTG), with respect to the structure and the reactionmechanism. For instance, BTG does not need calcium ion for expressingthe enzymatic function thereof. Due to this, differentiation in use ofthe BTG from the fish-derived transglutaminase to be obtained by thepresent invention would be necessary for the intended protein to beutilized.

As to the differences between BTG and the fish-derived transglutaminase,where the fish-derived transglutaminase is reacted with a substrate orwhere the reaction is desired to be stopped, the calcium ion dependencecould be utilized so as to control the reaction time and the reactivityof the enzyme. Also, the fish-derived transglutaminase is less stableagainst heat than BTG, so the fish-derived transglutaminase can bedenatured at relatively low temperature, which can be a merit forproduction of anti-heat food.

One method for obtaining a fish-derived DNA fragment containing a genewhich codes for a polypeptide possessing transglutaminase activityemploys a probe based on a synthesized DNA fragment coding for aminoacid residues near the active center of guinea pig transglutaminase. Anexample of such a DNA fragment is one which codes for the amino acidsequence of SEQ ID NO:2. Such probes are used for plaque hybridizationisolation of the desired DNA fragment from the cDNA prepared from tissueof fish. Prior to the present invention, the degree of homology betweenguinea pig transglutaminase and fish transglutaminase, as well asbetween their corresponding genes, was completely unknown. Thus, thesuccessful cloning of a DNA fragment encoding a fish transglutaminaseusing this method is unexpected.

Other methods of acquiring a fish-derived DNA fragment containing a genewhich codes for a polypeptide possessing transglutaminase activityinclude the following:

(A) Isolating and purifying a fish-derived polypeptide which possessestransglutaminase activity, determining the amino acid sequence andchemically synthesizing the corresponding base sequence encoding thedetermined amino acid sequence;

(B) Synthesizing a portion of the DNA sequence corresponding to thedetermined amino acid sequence, and hybridizing or "probing" thesynthesized portion of DNA with a cDNA bank or genomic DNA bank of thefish with the synthesized portion. Cloning may then be performed by thehybridization method or the polymerase chain reaction (PCR) method; and

(C) Translating mRNA transcribed from cDNA in an in vitro wheat germ orrabbit reticulocyte translation system, determining the section of mRNAcorresponding to a polypeptide which possesses transglutaminaseactivity, and cloning the corresponding cDNA fragment.

Examples of DNA fragments having the gene which codes for a fish-derivedpolypeptide possessing transglutaminase activity according to thepresent invention include those coding for a polypeptide selected fromthe group consisting of SEQ ID NOS:4, 6, 8, 10, 31, 33, 70 and 72.

The DNA fragment may include a variety of different base sequences, fromthe point of view of the degeneracy of the genetic codon. Appropriatebase sequences may be easily selected and prepared by one skilled in theart, depending on the various elements of the gene expression system.

For example, a particular codon may be preferred over a degeneratecodon, depending on the nature of the host cell, or to avoid theformation of a secondary structure in the transcribed RNA, etc. Suchconsiderations may be, and preferably are, taken into account in thepreparation of a DNA fragment suitable for the present invention.

The present DNA fragment may result from cloning a natural occurringentity, or may be chemically synthesized DNA. The DNA fragment of thepresent invention may also have a naturally occurring or artificialsubstitution, deletion, insertion or inversion of one or more bases inthe base sequence.

The present DNA fragment includes mutants having substitution, deletionor insertion of base sequences on the basis of the difference in theindividualities of fishes and of the difference in the respective organsand tissues of them. The present DNA fragment has a multiplicity derivedfrom mult-copies of gene-dosage, for example, which may be a pseudogene.However, such still contain an essentially equivalent DNA fragmentcapable of expressing the transglutaminase activity. The presence ofthem is described in the following examples.

Concrete examples of DNA fragments suitable for the present inventioninclude SEQ ID NOS:3, 5, 7, 9, 28, 30, 32, 69 and 71, which coincidewith a sequence from a natural source (see Examples 1, 2 and 6 below).Further examples of the DNA fragment of the present invention includethose having a sequence which encodes a polypeptide selected from thegroup consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:70 and SEQ ID NO:72, and thoseencoding a protein having SEQ ID NO:20, one of SEQ ID NOS:34-45 or acombination thereof, one of SEQ ID NOS:46-51 or a combination thereof,or one of SEQ ID NOS:52-68 or a combination thereof.

The present invention may provide the desired enzyme through expressionof a fish-derived transglutaminase gene in a microorganism transformedby recombinant genetic technology. A recombinant plasmid useful in thepresent invention may be prepared by insertion of a DNA fragment havingthe fish-derived gene which codes for a polypeptide possessingtransglutaminase into a publicly-known or commercially-availableexpression vector corresponding to the desired expression system, usinga publicly-known, conventional method.

Suitable methods are generally described by Maniatis et al ("MolecularCloning," Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.(1982)) and Sambrook et al, ("Molecular Cloning: A Laboratory Manual,"Cold Spring Harbor Laboratory, Cold Spring Harbor Press, (1989)), andare adapted to correspond to the selected expression system. Expressionvectors for E. coli include plasmids which express a fused proteincomposed of T7gene1O, a linker peptide and a desired protein (morespecifically, the XPRESS SYSTEM™, manufactured by Invitrogen Co.), andplasmids which express a fused protein composed ofglutathione-S-transferase and a desired protein (more specifically, thepGEX-2T or pGEX-3T vector plasmid, manufactured by Pharmacia LKB Co.).More preferable expression vectors are pBSF2-SD7 and pT13sNco.

An example of an expression vector for yeast of the genus Saccharomycesis pYES2 (available from Invitrogen Co.), which may use a GAL1 promoterfrom the gene which codes for galactokinase, for the expression of aforeign gene.

The present invention also relates to transformants obtained byintroduction of a recombinant plasmid which carries the transglutaminasegene. Some organisms which can be used for the host of transformationinclude procaryotic cells such as E. coli, Bacillus subtilis, etc. aswell as eucaryotic cells such as yeast, fungus, etc. Thus, a way hasbeen found for the efficient mass production of fish-derived TGase.

The host organism for transformation is preferably E. coli or yeast,more preferably the E. coli HB101 or Saccharomyces cerevisiae INVSC2strain. The transformants of the present invention are capable ofproducing and accumulating the fish-derived transglutaminase enzyme inthe cells or in the culture medium by the expressing therecombinantly-introduced transglutaminase gene.

Finally, the present invention relates to a method for the production ofa polypeptide possessing transglutaminase activity, by culturing theabove-mentioned transformant. The culturing conditions may be determinedas deemed appropriate by one skilled in the art depending on the type ofhost used. Also, the expressed enzyme, which is accumulated in the cellsor secreted into the culture medium or both, may be isolated andpurified using any one or a combination of publicly-known, conventionalmethods.

Concretely, in a purification process similar to that applied to naturaltransglutaminase from the tissues, this recombinant transglutaminase(s)can be purified from the crude extracts. It has not been reported thattransglutaminase derived from fish possess saccharide chains. Thefish-derived transglutaminase produced in E. coli may not be added withany saccharide chains, but the transglutaminase produced in yeast may beadded with some saccharide chains in accordance with the function of theglycosylation mechanism in yeast cells.

Other features of the present invention will become apparent in thecourse of the following descriptions of exemplary embodiments which aregiven for illustration of the invention, and are not intended to belimiting thereof.

EXAMPLES

1. A DNA fragment having a gene which codes for transglutaminase fromPagrus major

Using a polytron and a teflon homogenizer, 1.3 g of the liver fromPagrus major was crushed in a solution (20 ml) of 4M guanidinethiocyanate and 1% beta-mercapto-ethanol. After 0.5% sodium laurylsarcosinate was added to and dissolved in the resulting cell suspension,the obtained solution was passed through a 23 gauge hypodermic needle 10times to cut up the chromosomal DNA. Next, the solution was centrifugedat 4° C. 5000 rpm for 20 minutes and the supernatant obtained. The totalRNA was further purified by conventional CsCl density gradientcentrifugation of the supernatant (Sambrook et al, "Molecular Cloning: ALaboratory Manual," Cold Spring Harbor Laboratory, Cold Spring HarborPress, 1989). The total amount of RNA obtained was 3.8 mg.

Of this total RNA, 1.3 mg was supplied to an mRNA purifying kit(obtained from Clontech) using an oligo (dT)-cellulose column, andapproximately 20 μg of purified mRNA molecules were obtained.

Of the obtained mRNA, 8 μg were used as a template for cDNA preparation.A random primer was used for cDNA synthesis, and a You-prime cDNAsynthesis kit (obtained from Pharmacia) was used to synthesizedouble-stranded cDNA. The obtained cDNA was then incorporated into aλ-phage vectro-λZapII (obtained from Stratagene) at its restrictionenzyme site EcoRI, after which a GIGAPACK II GOLD (obtained fromStratagene) packaging kit was used to prepare and acquire the Pagrusmajor cDNA library incorporated in the phage. The titer of this librarywas 1.2×10⁶ pfu/μg vector.

A sample of host E. coli XL1-Blue cells were infected with 6.0×10⁴ pfuof phage from the above Pagrus major cDNA library, after which theinfected cells were spread at 1.5×10⁴ pfu per plate onto four 150 mmdiameter agar plates. The cells were cultured at 37° C. for about 9.5hours, and then the phage plaques formed on the plates were transferredonto a nylon membrane (HIBOND-N, manufactured by Amersham). Next, thenylon membrane was treated with an alkali to denature the DNA,neutralized and washed. Then, the membrane was treated at 80° C. for 3hours to immobilize the DNA onto the membrane.

Prehybridization of the prepared nylon membrane was then effected at 42°C. for 2 hours, followed by hybridization at 42° C. for 16 hours. Thecomposition of the prehybridization solution was 6× SSC (composition of1× SSC: 0.15M NaCl, 0.015M sodium citrate, pH 7.0), 5× Denhardt'ssolution (composition of 1× Denhardt's solution: 0.02% BSA, 0.02%Ficoll, 0.02% polyvinyl pyrrolidone), 20% formamide, 100 μg/ml ofherring testis DNA and 0.1% SDS. Also, the DNA probe used for thehybridization was a synthesized DNA fragment(5'-GTCAAGTACGGCCAGTGCTGGGTCTTCGC-3', SEQ ID NO:11; Ikura et al,Biochemistry, 27, 2898-2905, 1988) encoding amino acid residues locatednear the active center of guinea pig transglutaminase, labeled at the 5'terminal end with gamma-³² P! ATP. The candidate strains for thepositive clones obtained by this screening were further subjected to Asecond and third screening to finally obtain 4 positive clones.

The each infected cell maintaining the above-mentioned respective 4positive cDNA clones were then infected with helper phage (R408) totransform the cDNA derived from each of the positive clones intophagemid vector pBluescriptSK(-). The length of the inserted cDNA ofeach of the 4 clones was 0.5 kbp (kilobase pairs), 1.5 kbp, 2.5 kbp and1.0 kbp, and the clones were named pSLTG2, pSLTG4, pSLTG5 and pSLTG6,respectively.

Next, a restriction enzyme map for each of the cDNA clones was made, andSouthern blotting analysis was done with the inserted cDNA of pSLTG5 asthe probe. As it was made clear that pSLTG5 (with inserted cDNA length2.5 kbp) included the other 3 cDNA clones, the DNA base sequence for theinserted cDNA of pSLTG5 was determined. Analysis of the base sequencewas done with a Sequenase Version 2.0 (available from U.S.B. Co.) kit,according to conventional methods. The results showed a DNA sequencecontaining 2520 base pairs (SEQ ID NO:5). Also, the restriction enzymemap of pSLTG5 is shown in FIG. 1.

The DNA sequence contained a segment which showed a very high degree ofhomology with the DNA probe used (SEQ ID NO:11). The amino acid sequencededuced from the base sequence of the inserted cDNA of pSLTG5corresponds to SEQ ID NO:4. This amino acid sequence includes an activecenter sequence of 8 amino acid residues,Tyr-Gly-Gln-Cys-Trp-Val-Phe-Ala (SEQ ID NO:2) (Nakanishi et al, Eur. J.Biochem., 202, 15-21, 1991) which is present both in transglutaminasederived from guinea pig liver and in human blood clotting factor XIII.The E. coli strain (AJ 12673) transformed with the DNA plasmid pSLTG5which contains the Pagrus major transglutaminase cDNA obtained asdescribed above, Escherichia coli XL1-Blue/pSLTG5, is deposited with theFermentation Research Institute, Agency of Industrial Science andTechnology (1-3, Higashi, 1-chome, Tsukuba-shi, Ibaraki-ken 305, Japan)under the deposit number FERM BP-4114.

2. A DNA fragment having a gene which codes for transglutaminase fromTheragra chalcogramma (Alaska pollack)

Using a polytron and a teflon homogenizer, 2.3 g of Alaskan pollackliver were crushed in a solution (20 ml) of 4M guanidine thiocyanate and1% beta-mercaptoethanol. After 0.5% sodium lauryl sarcosinate was addedto and dissolved in the cell suspension, the resulting solution waspassed through a 23 gauge hypodermic needle 10 times to cut up thechromosomal DNA. Next, the solution was centrifuged at 4° C., 10,000 rpmfor 20 minutes and the supernatant obtained. The total RNA was furtherpurified by conventional CsCl density-gradient centrifugation of thesupernatant (Sambrook et al, "Molecular Cloning: A Laboratory Manual,"Cold Spring Harbor Laboratory, Cold Spring Harbor Press, 1989). Thetotal amount of RNA obtained was 7.2 mg. Of this total RNA, 2.3 mg wassubjected to an mRNA purifying kit (available from Clontech) using anoligo (dT)-cellulose column, and approximately 23 μg of purified mRNAmolecules were obtained.

Of the obtained mRNA, 4 μg were used as a template for cDNA synthesis. Arandom primer was used for DNA synthesis, and a You-prime cDNA synthesiskit (available from Pharmacia) was used to synthesize double-strandedcDNA. The obtained cDNA was then incorporated into lambda-phagevector-lambda ZapII (available from Stratagene) at its restrictionenzyme cutting site EcoRI, after which a GIGAPACK II GOLD (availablefrom Stratagene) packaging kit was used to produce and acquire an Alaskapollack cDNA library, incorporated in the phage. The titer of thelibrary was 4.1×10⁵ pfu/μg vector.

A sample of host E. coli XL1-Blue cells were infected with 5.8×10⁴ pfuof phage from the Alaska pollack cDNA library, after which it was spreadat 1.5×10⁴ pfu per plate onto 4 agar plates of 150 mm diameter. Thecells were cultured at 37° C. for about 9.5 hours, and then the phageplaques formed on the plates were transferred onto a nylon membrane(HIBOND-N, manufactured by Amersham). Next, the nylon membrane wastreated with an alkali to denature the DNA, neutralized and washed.Then, the membrane was treated at 80° C. for 3 hours to immobilize theDNA onto the membrane.

Prehybridization of the prepared nylon membrane was then effected at 42°C. for 2 hours, followed by hybridization at 42° C. for 16 hours. Thecomposition of the prehybridization solution was 6× SSC (composition of1× SSC: 0.15M NaCl, 0.015M sodium citrate, pH 7.0), 5× Denhardt'ssolution (composition of 1×Denhardt's solution: 0.02% BSA, 0.02% Ficoll,0.02% polyvinyl pyrrolidone), 20% formamide, 100 μg/ml of herring testisDNA and 0.1% SDS. A DNA probe to be used for the hybridization was basedon a DNA fragment of approximately 300 base pairs which may code for theamino acid sequence of the region near the active center, which could becut off from Pagrus major liver transglutaminase cDNA. The DNA fragmentwas obtained from Pagrus major liver cDNA using restriction enzymes ClaIand BamHI, followed by random labelling with α-³² P! dCTP. The candidatestrains for the positive clones obtained by this screening were furthersubjected to second and the third screenings to finally obtain 8positive clones.

The each infected cell possessing the above-mentioned respective 8positive cDNA clones was then infected with helper phage (R408) totransfer the cDNA derived from each of the positive clones into phagemidvector pBluescript SK(-). The eight clones were named pALTG1, pALTG2,pALTG3, pALTG6, pALTG7, pALTG8, pALTG9 and pALTG10, respectively. Ofthese, the lengths of the inserted cDNA of pALTG1, pALTG3, pALTG6 andpALTG8 were verified, a restriction enzyme map was made, and the cDNAbase sequences were analyzed at the 5' end and the 3' end to find thecorrelation between each clone, shown in FIG. 2. Here, a FluorescentPrimer Cycle Sequencing Kit (manufactured by A.B.I. Co.) was used foranalysis of the base sequences.

Next, synthetic primers (20 bases) for the sequencing were prepared,based on a portion of the base sequence of the obtained cDNA, todetermine the entire base sequence of the inserted cDNA of pALTG8.Analysis of the base sequence was done by conventional methods, using aSequenase Version 2.0 (available from U.S.B. Co.) kit. The resultsshowed a DNA sequence of 2921 base pairs (SEQ ID NO:9). The amino acidsequence deduced from SEQ ID NO:9 corresponds to SEQ ID NO:8. This aminoacid sequence includes an active center sequence of 8 amino acidresidues, Tyr-Gly-Gln-Cys-Trp-Val-Phe-Ala (SEQ ID NO:2), which ispresent both in transglutaminase derived from guinea pig liver and inhuman blood clotting factor XIII. The E. coli strain (AJ12709)transformed with the DNA plasmid pALTG8, containing the Alaska pollacktransglutaminase cDNA obtained as described above, Escherichia coliXLI-Blue/pALTG8, is deposited with the Fermentation Research Institute,Agency of Industrial Science and Technology (1-3, Higashi, 1-chome,Tsukuba-shi, Ibaraki-ken 305, Japan) under the deposit number FERMBP-4115.

Alaska pollack-derived transglutaminase of expressing in other tissuesthan liver has also been investigated. For instance, cloning of atransglutaminase cDNA from Alaska Pollack muscular tissue has beenattempted.

11.5 g of Alaska Pollack muscular tissue were ground in 80 ml of an 1%beta-mercaptoethanol solution of 4M guanidine thiocyanate, usingpolytron and teflon homogenizer. 0.5% sodium lauryl sarcocinate wasadded to and dissolved in the resulting cell suspension, and thesolution was passed through a 23-gauge hypodermic needle seven times andsuccessively through a 25-gauge injection needle seven times, wherebythe chromosomal DNA was fragmented. Next, the solution was subjected tocentrifugation of 10,000 rpm for 20 minutes at 4° C. or lower, and thesupernatant was collected. From the supernatant, a complete RNA waspurified by CsCl density gradient centrifugation of a conventionalmanner (refer to Sambrook et al, "Molecular Cloning: A LaboratoryManual," Cold Spring Harbor Laboratory, Cold Spring Harbor Press(1989)). The amount of the thus obtained complete RNA was 2.1 mg. 1.7 mgof it was applied to an mRNA purifying kit (Clontech) using anoligo(dT)-cellulose column, to purify the mRNA molecule, the yield ofwhich was about 21 micrograms.

Of the thus obtained mRNA, 3.2 micrograms were used as a template forsynthesis of cDNA. For synthesis of cDNA, a random primer was used, anda Time Saver cDNA synthesis kit (Pharmacia) for preparing adouble-stranded cDNA was used.

Plaque hybridization using the above cDNA library, was effected in thesame manner as for the DNA fragment coding for transglutaminase derivedfrom Alaska pollack liver, but no positive cDNA clone could be obtained.Therefore, a cDNA fragment was obtained by the method which will bementioned below in detail.

The oligonucleotides as synthesized on the basis of the gene basesequence of Alaska pollack liver-derived transglutaminase, using theprepared Alaska pollack muscular cDNA group as a template, were used asprimers, and a cDNA fragment of Alaska pollack muscular transglutaminasewas specifically amplified by a PCR method (polymerase chain reactionmethod) using Amplitaq DNA Polymerase (Takara Shuzo).

As shown in FIG. 10, Pr. 10 (5'-TTGGAAGCTTGTAAGAGCAACTCTTGGAAA-3'; SEQID NO:21) and Pr. 970 (5'-TTGTACACTCGATCGATGGAGAGGT-3'; SEQ ID NO:22)were both used as primers for synthesizing the cDNA fragment, for the 5'terminal region (the region coding for N-terminal region oftransglutaminase) in the expected Alaska pollack muscle-derivedtransglutaminase cDNA structure. After PCR, a DNA fragment of about 980bp was amplified. Next, the terminal of the fragment was made blunt andthen inserted into the restriction enzyme HincII cleaved site of pUC18vector.

For the gene amplification of the center region, Pr. 620(5'-TCTGCTTTGGGATCCTTGACCGCT-3'; SEQ ID NO:23) and Pr. 2000(5'-TGAAGGAGAGCTCCACAGACACA-3'; SEQ ID NO:24) were both used assynthetic oligonucleotide primers. Into these primers, were artificiallyinserted a restriction enzyme BamHI cleavage recognizing site and arestriction enzyme SacI cleavage recognizing site, respectively. AfterPCR reaction, the amplified DNA fragment of about 1.4 kbp was prepared,and this was digested with the preceding enzymes to give DNA fragments,which were inserted into the same restriction enzyme sites ofpBluescript IISK- to obtain cDNA clones.

Further, for amplification of the cDNA fragment in the 3' terminalregion (the region coding for C-terminal region of transglutaminase),the intended region was first amplified with PCR primers Pr. 10-1F(5'-ATGATGTCAAAGGCTGTCAC-3'; SEQ ID NO:25) and Pr. 8-1R(5'-TCTTACCATATAAGTTGTAA-3'; SEQ ID NO:26). However, since the primerscaused amplification of other small fragments than the intended DNAfragment, the thus amplified DNA group was again subjected to geneamplification, using the same primer Pr. 10-1F and a novel primer of Pr.3-2F2R (5'-ATTGATTAACAACAAAATGG-3'; SEQ ID NO:27) as templates. As aresult, a DNA fragment of about 800 bp was amplified. Both terminals ofthe present cDNA fragment were also made blunt in the same manner asmentioned above, and the resulting cDNA fragments were inserted into therestriction enzyme EcoRV site of pBluescript IISK-.

A plasmid having the cDNA fragment having each of the above-mentionedthree regions was applied to E. coli XL1-Blue for transformation,whereby N-terminal clones of Nos. N-3, N-4 and N-5, center region clonesof Nos. SB-4, SB-5, SB-21, SB-22, and SB-30 and C-terminal clones ofNos. C-6, C-9 and C-13 were obtained. Next, the base sequence of each ofthe above-mentioned eleven DNA clones was sequenced by a known methodusing Applied Biosystems' Tag dideoxy Terminator Cycle Sequencing Kit.As a result, two kinds of genes coding for transglutaminase were presentin Alaska pollack muscule, the one having DNA sequence as shown as SEQID NO:28 in the Sequence List and the other having DNA sequence as samestructure as the Alaska pollack liver-derived transglutaminase cDNA asshown as SEQ ID NO:7.

From the above, it was clarified that the transglutaminase of SEQ IDNO:7 is an Alaska pollack transglutaminase as expressed beyond the kindof the organ; and that the transglutaminase of SEQ ID NO:28, though notobtained as a cDNA of a complete length, was different from theliver-derived transglutaminase only in the point of a several-basesubstitution, a base deletion of 12 bp and a base insertion of 3 bp inthe structural gene. Thus, both genes were clarified to be highlyhomologous to each other.

An E. coli strain (AJ 12790) of E. coli XLI-Blue/N3 having plasmid N3containing a part of the Alaska pollack muscle-derived transglutaminasecDNA fragment (SEQ ID NO:28) obtained in the above manner has beendeposited in Fermentation Research Institute of Japan as FRI DepositionNo. 4147 (FERM BP-4147); an E. coli strain (AJ 12791) of E. coliXLI-Blue/N5 having plasmid N5 as FRI Deposition No. 4148 (FERM BP-4148);an E. coli strain (AJ 12792) of E. coli XLI-Blue/SB4 having plasmid SB4as FRI Deposition No. 4149 (FERM BP-4149); an E. coli strain (AJ 12793)of E. coli XLI-Blue/SB5 having plasmid SB5 as FRI Deposition No. 4150(FERM BP-BP-4150); an E. coli strain (AJ 12794) of E. coli XLI-Blue/SB21having plasmid SB21 as FRI Deposition No. 4151 (FERM BP-4151); and an E.coli strain (AJ 12795) of E. coli XLI-Blue/SB22 having plasmid SB22 asFRI Deposition No. 4152 (FERM BP-4152).

3. Construction of plasmid pIL6TG1 which express the Pagrus majortransglutaminase gene, its introduction into E. coli, and verificationof physiological activity of a TGase produced by the transformant

Plasmid pSLTG5 having the Pagrus major transglutaminase gene (cDNA)prepared in the above example was digested with restriction enzymes XbaIand EcoRV, as shown in FIG. 3, and a DNA fragment containing thetransglutaminase cDNA was obtained. In a separate procedure, expressionvector pBSF2-SD7 possessing a tryptophan promoter and trpA terminatorwas digested with BamHI. The DNA cutting end was then made flat withKlenow enzyme, then treated with XbaI to obtain a large DNA fragmentpossessing a tryptophan promoter. Expression plasmid pBSF2-SD7 is theplasmid listed in Bio/Technology, 8, pp. 1036-1040, 1990.

The two DNA fragments obtained by the above-described treatment wereligated together with T4 DNA ligase to obtain the Pagrus majortransglutaminase cDNA expression plasmid pIL6TG1. Verification of theDNA base sequence of this pIL6TG1 showed that one GC base pair wasmissing at the BamHI cleavage site, making it clear that the EcoRV sitewas present upstream from the trpA terminator in the plasmid. Aconventional method was used to introduce pIL6TG1 into E. coli HB101,and a transformant, Escherichia coli HB101/pIL6TG1, (AJ12730) wasprepared. AJ12730 is deposited with the Fermentation Research Institute,Agency of Industrial Science and Technology (1-3, Higashi, 1-chome,Tsukuba-shi, Ibaraki-ken 305, Japan) under the deposit number FERMBP-4116.

A colony of the acquired transformant was applied onto an agar platecontaining 200 μg/ml of ampicillin, and cultured at 30° C. overnight,after which approximately 2 cm² of the lawn of growing cells from theplate were inoculated into a Sakaguchi flask containing 100 ml of an M9casamino acid culture medium supplemented with 2% glucose, 200 μg/ml ofleucine, 200 μg/ml of proline, 2.0 μg/ml of thiamine-HCl, and 200 μg/mlof ampicillin. The mixture was cultured at 30° C. for about 16 hours tocollect the cells.

To the collected cells were added 0.3 ml of a 0.5M EDTA solution, and 30ml of a mixture of 20 mm Tris-HCl and 30 mM NaCl to prepare asuspension. Further, 1 ml of a 4 mg/ml lysozyme solution was addedthereto, the mixture was stirred and then allowed to stand at 0° C. for1 hours. After this, the cell suspension was subjected to ultrasoniccrushing, and the ultrasonically crushed cells were centrifuged (8000rpm for 10 minutes) to prepare a supernatant. Also, a separatesupernatant was prepared from centrifuging crushed cells of E. coliHB101/pBSF2-SD7, possessing a pBSF2-SD7 plasmid with no transglutaminasecDNA, transformed in the same manner as E. coli HB101/pIL6TG1 (AJ12730).

The transglutaminase activity of each of the supernatants was determinedaccording to an activity detection method which measures the change influorescence intensity (fluorescence intensity at 480 nm, resulting fromexcitation irradiation with 350 nm wavelength light) due to bonding ofmonodansyl cadaverine with dimethylated casein. This activity detectionmethod is based on the method described in Nippon Suisan Gakkaishi(1991), Vol. 57, pp. 1203-1210, with a few modifications. That is, 150μl of each of the test samples was added to a solution (adjusted to 2.5ml after addition of the samples) composed of 1 mg/ml of dimethylatedcasein, 15 μM of monodansyl cadaverine, 5 mM of CaCl₂, 50 mM of Tris-HCl(pH 7.5) and 3 mM of dithiothreitol (DTT), which was stirred and kept at37° C. for 30 minutes. After the reaction, EDTA solution was added to afinal concentration of 10 mM, and the fluorescence intensity of each ofthe reaction solutions was measured using a fluorescence intensity meter(available from Shimazu RF-520).

The results, which can be seen in Table 1, clearly show thattransglutaminase activity was present in the cell extract of E. coliwhich possesses an expression plasmid having transglutaminase cDNA,obtained in the example described above. It became obvious from thisresult that the cDNA we acquired coded for transglutaminase.

                  TABLE 1    ______________________________________                            Relative fluorescence    Strain        TG cDNA   intensity    ______________________________________    pBSF2-SD7/HB101                  absent    9    pIL6TG1/HB101 present   257    ______________________________________     Note: TG cDNA = transglutaminase cDNA

4. Construction of plasmid pTTG2-22, which expresses the Pagrus majortransglutaminase gene, its introduction into E. coli and theverification of physiological activity of the TGase produced by thetransformant

Next, in order to further produce a large amount of transglutaminase, wemade an improvement in the transglutaminase expression plasmid. Anexplanation thereof is given below.

Since the amount of transglutaminase expressed by transglutaminaseexpression plasmid pIL6TG1 obtained in Example 3 was small, furtherdiligent research was conducted in order to construct a plasmid capableof expressing more transglutaminase than pIL6TG1, and a modification wasmade to increase the translation efficiency of the transglutaminase genein E. coli.

The modification involved substituting a portion of the DNA basesequence of the natural transglutaminase gene with chemicallysynthesized DNA to alter the base sequence without changing the codedamino acid sequence, and designing it so that the transglutaminase genemight yield a more efficient expression in E. coli.

As shown in FIG. 4, chemically synthesized DNA fragments (SEQ IDNOS:12-19) were designed and prepared, which included a consensus SD(Shine-Dalgarno) sequence (5'-TAAGGAGGT-3') and a region coding for thesection of the transglutaminase of SEQ ID NO:6 from the amino-terminalmethionine to the 32nd amino acid (leucine), downstream therefrom, withthe intention of incorporating them into the natural transglutaminasegene, according to the procedure described below. Codons preferred in E.coli and/or codons containing AT rich sequences were selected for theregion coding for the section of transglutaminase from theamino-terminal methionine to ³² Leu.

Eight DNA oligomers (SEQ ID NOS:12-19) for construction of thechemically synthesized DNA fragment named DNA-1 (SEQ ID NO:1) weresynthesized with a DNA synthesizer (manufactured by A.B.I.). Theseoligomers were combined and linked using a conventional method,annealing and ligating with T4 DNA ligase, to prepare the chemicallysynthesized DNA-1. Next, pUC19 was digested with the restriction enzymesEcoRI and HindIII, and the DNA fragments were cloned therein toconstruct pFTGN6 (see FIG. 5). This plasmid was also used forverification of the DNA base sequence, confirming that the prepared DNAhad the intended sequence.

Plasmid pFTGN6 was digested with the restriction enzymes ClaI andHindIII, and was further treated with restriction enzyme HaeII, to yieldDNA fragment C (approximately 110 base pairs) having ClaI and HaeIIcleavage termini (see FIG. 6).

In a separate procedure, plasmid pSLTG5, which includes cDNA coding forPagrus major transglutaminase, was treated with EcoRI, HaeII and NcoI toobtain a DNA fragment (DNA fragment B) of approximately 1.44 kbp(kilobase pairs), which contained the major part of transglutaminasecDNA (see FIG. 6).

Expression vector pT13sNco (listed in J. Biochem., 104, 30-34, 1988),possessing a tryptophan promoter and trpA terminator, was digested withNcoI and BamHI, and both of the cleavage termini were made flat withKlenow enzyme, thus acquiring a large DNA fragment. This was furtherself-ligated with T4 DNA ligase to obtain the plasmid pTTNco. Next,pTTNco was digested with ClaI and NcoI, after which the cleavage terminiwere dephosphorylated with alkaline phosphatase, to prepare a large DNAfragment (DNA fragment A) possessing a tryptophan promoter and a trpAterminator (see FIG. 6).

Each of the DNA fragments A, B and C was ligated with T4 DNA ligase, toconstruct plasmid pTTG1 (see FIG. 7). For greater clarity, DNA fragmentB, represented by an outlined bar in FIG. 6, is represented by a solidbar in FIGS. 7 and 8.

Next, the constructed plasmid pTTG1 was treated with BamHI, the cleavedtermini were made flat with Klenow enzyme, then it was digested withSacI to obtain the large DNA fragment shown in FIG. 8. Separately,pSLTG5 was treated with the restriction enzymes SacI and EcoRV toprepare a small DNA fragment. These DNA fragments were ligated with T4DNA ligase, to construct expression plasmid pTTG2-22 (see FIG. 8).Expression plasmid pTTG2-22 was then introduced into E. coli HB101 usinga conventional method to prepare a transformant, Escherichia coliHB101/pTTG2-22 (AJ12742). AJ12742 is deposited with the FermentationResearch Institute, Agency of Industrial Science and Technology (1-3,Higashi, 1-chome, Tsukuba-shi, Ibaraki-ken 305, Japan) under the depositnumber FERM BP-4117.

A colony of the acquired transformant was applied onto an agar platecontaining 200 μg/ml of ampicillin, and cultured at 30° C. overnight,after which approximately 2 cm² of the lawn of growing cells from theplate were inoculated into a Sakaguchi flask containing 100 ml of an M9casamino acid culture medium supplemented with 2% glucose, 200 μg/ml ofleucine, 200 μg/ml of proline, 2.0 μg/ml of thiamine-HCl, and 200 μg/mlof ampicillin. The mixture was culture at 30° C. for about 16 hours tocollect the cells.

A supernatant from crushed cells was prepared from the collected cellsin the same manner as in Example 3. Also, another supernatant wasprepared by centrifugation of crushed E. coli cells possessing a plasmidwithout transglutaminase cDNA (E. coli HB101/pTTNco) in the same manner.

The transglutaminase activity of each of the supernatants (50 μl) wasverified in the same manner as in Example 3, according to an activitydetection method which measures the change in fluorescence intensity(fluorescence intensity at 480 nm, excited with 350 nm wavelength light)due to bonding of monodansyl cadaverine with dimethylated casein. Theresults in Table 2 clearly show that transglutaminase activity waspresent in the cell extract of E. coli which possesses an expressionplasmid (pTTG2-22) having transglutaminase cDNA, obtained in the exampledescribed above. This result showed that the cDNA we acquired codes fortransglutaminase.

                  TABLE 2    ______________________________________                            Relative fluorescence    Strain        TG cDNA   intensity    ______________________________________    pTTNco/HB101  absent    21    pTTG2-22/HB101                  present   910    ______________________________________     Note: TG cDNA = transglutaminase cDNA

Next, it was verified whether or not the above-mentioned cell extractsolution could gelatinize the myosin B solution derived from Alaskapollack (Theragra chalcogramma). 1.0 ml of an Alaska pollack-derivedmyosin B solution (approx. 6 mg/ml) was used as the substrate, and foursamples were prepared in which the following components were added:

(1) 470 μl of 50 mM CaCl₂ and 100 μl of E. coli raw extract solutionexhibiting transglutaminase activity was detected;

(2) 470 μl of 50 mM CaCl₂ ;

(3) 100 μl of E. coli raw extract solution exhibiting transglutaminaseactivity; and

(4) nothing.

Each of the samples were prepared in a test tube, and the mixtures werestirred and allowed to stand at room temperature for about 16 hours. Thegelation of myosin B was judged by inverting the test tube and observingwhether the reaction contents dripped (were able to flow at ambienttemperature) or were solidified (hardened). The results, shown in Table3 below, indicated gelation only in the sample containing both calciumchloride and the E. coli extract which exhibited transglutaminaseactivity, proving that the cDNA acquired according to the presentinvention contained a DNA fragment which codes for transglutaminase.

                  TABLE 3    ______________________________________           TG added      TG not added             Ca present                       Ca absent Ca present                                         Ca absent    ______________________________________    Gelation of             +         -         -       -    myosin B    ______________________________________     Note:     TG: cell extract exhibiting transglutaminase activity     Ca: calcium chloride     +: gelatinized     -: not gelatinized

5. Construction of plasmid pYSTG1, which expresses the Pagrus majortransglutaminase gene, its introduction into S. cerevisiae, and theverification of physiological TGase activity produced by thetransformant

Plasmid pSLTG5, containing cDNA which codes for Pagrus majortransglutaminase, was treated with the restriction enzymes EcoRI andXbaI to obtain a DNA fragment of approximately 2.5 kbp (kilobase pairs)having transglutaminase cDNA.

In a separate procedure, pYES2 (available from Invitrogen Co.), whichuses a GAL1 promoter (GAL1 represents a gene which codes forgalactokinase) for the expression of foreign genes, was used as anexpression vector for S. cerevisiae. pYES2 was treated with the samerestriction enzymes EcoRI and XbaI, to prepare a DNA fragment ofapproximately 5.8 kbp.

The two above-mentioned DNA fragments were ligated with T4 DNA ligaseaccording to a conventional method, and the product was introduced intoa competent E. coli HB101 cell (available from Takara Shuzo Co.)according to a conventional method. Selection of the transformant wasdone using an L-agar plate containing the antibiotic ampicillin at aconcentration of 100 μg/ml. Next, six individual strains were picked upfrom the cultured E. coli colonies using a sterilized toothpick, andeach was transferred to a separate 3 ml L-culture medium containing 100μg/ml of ampicillin. Each sample was shake-cultured at 30° C. for 16hours.

Plasmid DNA was prepared from 2 ml of each of the cultures using thealkaline SDS method, and the pattern of restriction enzyme cleavage wasanalyzed by agarose gel electrophoresis. The analysis indicated thatplasmids obtained from four of the strains were the desired one, and theplasmid was named pYSTG1. Further, the DNA base sequence was analyzedfor the junction regions at the time of construction of the plasmid,verifying that the base sequences were correct.

The pYSTG1, constructed as described above, was then introduced into ayeast, Saccharomyces cerevisiae, INVSC2 strain (MATα, his3-Δ200,ura3-167), according to a conventional yeast transformation method,further using a conventional alkaline cation method kit (manufactured byBio101, purchased from Funakoshi, Inc.). A YNB culture medium(composition: 0.67% yeast nitrogen base without amino acids (availablefrom Difco Co.), 2% glucose, 2% agar) containing 20 mg/l of histidinewas used as the selection plate. In this manner a transformant,Saccharomyces cerevisiae INVSC2/pYSTG1 (AJ14679) was obtained. AJ14679is deposited with the Fermentation Research Institute, Agency ofIndustrial Science and Technology (1-3, Higashi, 1-chome, Tsukuba-shi,Ibaraki-ken 305, Japan) under the deposit number FERM BP-4085.

The transformant yeast, grown on the above-mentioned YNB culture mediumplate containing 20 mg/l of histidine, was inoculated into 100 ml of aYPD culture medium (composition: 1% yeast extract, 2% bactopeptone, 2%glucose), and the mixture was cultured at 30° C. for a day and a nightwith shaking. The culture was then centrifuged to collect the cells,which were then washed with 1% yeast extract, after which they weresuspended in 10 ml of the same solution. Next, 5 ml of the suspensionwas added to 95 ml of a culture medium composed of 1% yeast extract, 2%bactopeptone and 2% galactose, and this mixture was cultured at 30° C.for 18 hours with shaking. Recombinant yeast was cultured in agalactose-containing culture medium to induce transcription from GAL1promoter. Also, the remaining 5 ml of the suspension mentioned above wasinoculated in the same manner into 95 ml of a liquid YPD culture medium,and culturing was done under conditions which did not inducetranscription from GAL1 promoter.

In a separate procedure, a host yeast INVSC2 strain not possessing thepYSTG1 plasmid was grown on a separate YPD culture plate, and inoculatedin the same manner described above into 100 ml of a liquid YPD culturemedium. The mixture was cultured at 30° C. The above-describedprocedures were then followed, and 5 ml of the obtained suspension wasinoculated into 95 ml of the liquid YPD culture medium and cultured.

Preparation of the sample for measurement of transglutaminase activitywas done in the following manner. Yeast, which had been cultured in 100ml of a culture medium, were centrifuged to collect the cells, to which15 ml of a 20 mM Tris-HCl buffer solution (pH 7.5) containing 30 mMsodium chloride were added to suspend the cells. To the suspension, 0.25ml of 0.5M EDTA was added, the mixture was then stirred, after which analmost equal volume of glass beads (diameter=0.75 mm) were added to thesuspension. The mixture was stirred vigorously for 4 minutes. Afterthis, centrifugation was done at 6000 rpm for 10 minutes, the insolubleswere removed, and the supernatant thereof was used as a sample formeasurement of transglutaminase activity.

The transglutaminase activity of the supernatants (50 μl or 150 μl) fromeach of the disrupted cell solutions was verified in the same manner asin Example 3 described above, according to an activity detection methodwhich measures the change in fluorescence intensity (fluorescenceintensity at 480 nm in response to excitation with 350 nm wavelengthlight) due to bonding of monodansyl cadaverine with dimethylated casein(see Table 4). Here, the fluorescence intensity was treated as zero whenthe disrupted cell solution of yeast INVSC2 strain not containing thepYSTG1 plasmid was added to the solution for the activity measurement.This result clearly shows that transglutaminase activity was present inthe disrupted cell extract solution of the yeast which possessed theexpression plasmid pYSTG1, having transglutaminase cDNA as obtained inExample 1 described above, under the induced condition of transcriptionfrom the GAL1 promoter. Also, when EDTA was preadded to the enzymereaction solution and calcium ion was removed, there was, as expected,no detection of transglutaminase activity. Based on this result, thecDNA which we acquired was that which codes for calcium ion-dependanttransglutaminase.

                  TABLE 4    ______________________________________                            Tran-    Relative                    EDTA    scription                                     fluorescence    Assayed samples *1                    *2      induction                                     intensity    ______________________________________    Crushed cell solution 50 μl                    absent  no       65    Crushed cell solution 50 μl                    absent  yes      260    Crushed cell solution 150 μl                    absent  no       66    Crushed cell solution 150 μl                    absent  yes      348    Crushed cell solution 150 μl                    added   no       0    Crushed cell solution 150 μl                    added   yes      0    ______________________________________     *1 Crushed cell solution of yeast INVSC2 strain having expression plasmid     pYSTG1     *2 Before addition of the crushed cell solution, EDTA was added to the     transglutaminase activity measurement solution to a final concentration o     100 mM, or absent from the mixture

6. DNA fragment containing a gene which codes for transglutaminase offlounder

Using polytron and a teflon homogenizer, 1.5 g of the liver fromflounder was crushed in a solution (20 ml) of 4M guanidine thiocyanateand 1% beta-mercaptoethanol. After 0.5% sodium laurylsarcosinate wasadded to and dissolved in the resulting cell suspension, the obtainedsolution was passed through a 23-gauge hypodermic needle 8 times tofragment the chromosomal DNA. Next, the solution was centrifuged at 4°C. 1000 rpm for 20 minutes and the supernatant obtained. The completeRNA was further purified by a conventional method, through CsCl densitygradient centrifugation of the supernatant (Sambrook et al., MolecularCloning: a laboratory manual, Cold Spring Harbor Laboratory, Cold SpringHarbor Press (1988)). The amount of the complete RNA obtained was 4.7mg. Of this, 1.6 mg was subjected to an mRNA purifying kit (Clontech)using an oligo(dT)-cellulose column, and approximately 23 microgram of apurified mRNA molecule was obtained.

Of the obtained mRNA, 4.4 microgram was used as a template for cDNAsynthesis. A random primer was used for eDNA synthesis, and a Time SavercDNA synthesis kit (Pharmacia) was used to synthesize a double-strandedcDNA. The obtained cDNA was then inserted into a lambda-phagevector-lambdaZapII (Stratagene) at its restriction enzyme site EcoRI,after which a GIGAPACK II GOLD (Stratagene) packaging kit was used toprepare and acquire a cDNA library of flounder incorporated in the phageprotein. The titer of this library was 2.0×10⁵ pfu/microgram vector.

Host cells XLI-Blue were infected with 2.2×10⁵ pfu of phage from saidcDNA library of flounder, after which they were spread onto 11 agarplates of diameter 150 mm at 2×10⁴ pfu per plate. The cells werecultured at 37° C. for about 6 hours, and then the phage plaques formedon the plates were transferred on a nylon membrane (Hibond-N,manufactured by Amersham). Next, the thus transcribed nylon membrane wastreated with an alkali to denature the bound DNAs, neutralized andwashed. Then, the membrane was treated at 80° C. for 2.5 hours toimmobilize the DNA on the membrane.

Prehybridization of the obtained nylon membrane was then effected at 42°C. for 2 hours, followed by hybridization at 42° C. for 16 hours. Thecomposition of the prehybridization solution was 6× SSC (composition of1× SSC: 0.15M NaCl, 0.015M sodium citrate, pH 7.0), 5× Denhardt'ssolution (composition of 1× Denhardt's solution: 0.02% BSA, 0.02%Ficoll, 0.02% polyvinyl pyrrolidone), 20% formamide, 100 microgram/ml ofherring testis DNA and 0.1% SDS. Also, the DNA probe used for thehybridization was the DNA fragment of about 300 bp of a cDNA of Pagrusmajor liver transglutaminase (SEQ ID NO:3), the DNA fragment being ableto code for the region containing the amino acid residues near to theactive center and being able to be cleaved with restriction enzymes ClaIand BamHI and having been random-labeled with alpha-32P! dCTP. Thecandidate strains for the positive clones obtained by this screeningwere further subjected to second and third screening to finally obtain10 positive clones.

The infected cells maintaining the above mentioned 10 positive cloneswere then infected with helper phage (R408) to transform the cDNAderived from each of the positive clones into a form incorporated intophagemid vector pBluescriptSK(-). The length of the inserted cDNA ofeach of the 5 clones (named pFLTG10, 12, 16, 17, 21, respectively) ofthese clones was measured, from which the restriction enzyme cleavagemap was constructed and the cDNA base sequences at the 5' end and the 3'end were sequenced. For the base sequencing, a fluorescent primer cyclesequencing kit was used (A.B.I.). As a result, the pFLTG21 was found tobe a cDNA fragment of coding for the center part of a transglutaminasegene.

Next, for the purpose of obtaining a clone capable of coding for theC-terminal region of transglutaminase, which are not in the preceding 5clones, the above-mentioned flounder cDNA library was again screened inthe manner as mentioned below.

As a DNA probe for the screening, used were a DNA fragment of about 300bp capable of being cleaved out from clone pFLTG21 with restrictionenzyme EcoRI and a DNA fragment of about 500 bp capable of being cleavedout from clone pFLTG17 with restriction enzymes SalI and PstI, bothfragments being random-labeled with alpha-32P! dCTP. The otherconditions were same as those mentioned above. As a result, a positiveclone was obtained by the present screening, and finally 10 positiveclones were obtained after second and third screenings.

Of the preceding 10 positive clones, the vector of each of 4 cDNA cloneswas converted into pBluescript SK-, and the relationship between theinserted cDNA's was analyzed. The four clones were named pFLTG44, 51, 55and 63, respectively.

Further, for obtaining a clone capable of coding for the N terminalregion of transglutaminase, a flounder cDNA library was newly prepared,using a synthesized DNA primer as formed on the basis of the basesequence of the inserted eDNA 5' end of pFLTG12 (the primer was composedof 19 bases, having a sequence of 5'-ACACTGCCGGTCCATCGAA-3'; SEQ IDNO:29). As a template for synthesis of the cDNA, used was 2 microgram ofthe mRNA sample described above. The titer of the library obtained herewas 1.5×10⁴ pfu/microgram vector.

Next, host E. coli cells XL1-Blue were infected with 6×10³ pfu of phagefrom said cDNA library, and then the screening was carried out by themethod mentioned above. As the probe for the hybridization, used were aDNA fragment of about 300 bp capable of being cleaved from the precedingclone pFLTG21 with restriction enzyme EcoRI, and a DNA fragment of about500 bp capable of being cleaved from pFLTG17 with restriction enzymesSalI and PstI, both fragments being random-labeled with alpha-32P! dCTP.The candidate strains for the positive clones obtained by this screeningwere further subjected to second and third screening to finally obtainone positive cDNA clone.

The clone was converted into pBluescript SK- by the method mentionedabove, and the clone was named pFLTG60. Next, the length of the insertedcDNA of the clone was measured, the restriction enzyme map wasconstructed, and the cDNA base sequences of the 5' end and 3' endregions were sequenced.

Of the thus obtained and sequenced 10 cDNA clones, the base sequencefrom the restriction enzyme SalI site to the 3' end of the inserted cDNAin pFLTG17, and the base sequence of the inserted cDNA fragment in eachof pFLTG21, 55, 60 and 63 were sequenced. As a result, the DNA sequencesof SEQ ID NO:30 and SEQ ID NO:32 in the Sequence Listing were clarified.The difference between the two base sequences resides in the differencein the 1854th base. But the change of the base does not change thetranslated amino acid residue. The amino acid sequences to be translatedfrom the base sequences are shown as SEQ ID NOS:31 and 33. In addition,the relationship between the clones is shown in FIG. 11.

An E. coli strain (AJ 12798) of E. coli XLI-Blue/pFLTG21 having plasmidpFLTG21 containing a part of the flounder-derived transglutaminase cDNAfragment (SEQ ID NO:30) obtained in the manner as above has beendeposited in Fermentation Research Institute of Japan as FRI DepositionNo. 4154 (FERM BP-4154); an E. coli strain (AJ12799) of E. coliXLI-Blue/pFLTG55 having plasmid pFLTG55 as FRI Deposition No. 4155 (FERMBP-4155); an E. coli strain (AJ 12800) of E. coli XLI-Blue/pFLTG60having plasmid pFLTG60 as FRI Deposition No. 4156 (FERM BP-4156); an E.coli strain (AJ 12801) of E. coli XLI-Blue/pFLTG63 having plasmidpFLTG63 as FRI Deposition No. 4157 (FERM BP-4157); and an E. coli strain(AJ 12797) of E. coli XLI-Blue/pFLTG17S having plasmid pFLTG17S having acDNA clone fragment of the downstream region from restriction enzymeSalI of the cDNA fragment of plasmid pFLTG17 as FRI Deposition No. 4153(FERM BP-4153). Each plasmid having cDNA fragment coding fortransglutaminase derived from Paralichthys olivaceus are shown in FIG.12.

The respective cDNA fragments thus obtained in the manner as mentionedabove may easily be converted into one DNA fragment completelycontaining the coding region of transglutaminase by a known method.Plasmid pFLTG17S was digested with Pst I to prepare a large fragmentcontaining cDNA segment. Similarly, pFLTG63 was digested with Pst I toprepare a DNA fragment coding for the C-terminal region of thetransglutaminase. Both DNA fragments as mentioned above were ligatedwith T4 DNA ligase to construct pFLTG1-C in which the reading frame ofthe C-terminal region of transglutaminase was made to be conjugative.

On the other hand, pFLTG21 was digested with Bgl II and Pst I to preparea large fragment. Similarly, pFLTG60 was treated with Bgl II and Pst Ito prepare a DNA fragment coding for the N-terminal region of thetransglutaminase. Both DNA fragments as mentioned above were ligatedwith T4 DNA ligase to construct pFLTG1-N in which the reading frame ofthe N-terminal region of the transglutaminase was made to beconjugative.

After pFLTG1-N was digested with EcoR I, the vector DNA of which bothends were dephosphorylated was ligated to the cDNA fragment derived frompFLTG1-N, and then pFLTG2-N in which cDNA fragment was inserted into thevector in the opposite direction in contrast with the direction of cDNAfragment in pFLTG1-N. Then, pFLTG2-N was digested with Sal I to preparecDNA fragment coding for the N-terminal region of the transglutaminase.The cDNA fragment was incorporated into the restriction site Sal I inpFLTG1-C digested with Sal I to construct one cDNA fragment completelycontaining the coding region of the transglutaminase in the vector DNA.

On the other hand, transformation of E. coli strain recBC, sbcA with asingle-stranded plasmid DNA having two cDNA fragments each having aduplicated cDNA region (for example, cDNA fragments on the respectiveplasmids of pFLTG60 and pFLTG21; pFLTG21 and pFLTGI7; pFLTG17 andpFLTG63) at both ends brings about extremely easily recombination at theduplicated region in these cDNA fragments due to the recombinationmechanism in the host E. coli strain, resulting in construction of asuccessively continued cDNA fragment. Repeating the process, theintended cDNA of a complete length is obtained. Utilizing therecombination mechanism of the kind, various chimera genes areconstructed (Ogawa et al, Journal of Molecular Biology, vol. 226, pp.651-660 (1992)).

The translated amino acid sequence of each transglutaminase cDNA derivedfrom Pagrus major, Theragra chalogramma, and Paralichthys olivaceus wasalso investigated. As a result, some consensus amino acid sequences werefound among the amino acid sequences of transglutaminase derived fromfish. These consensus amino acid sequences are shown as SEQ ID NOS:34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44 and 45. Xaa indicates anonspecific amino acid residue.

7. Investigation as to whether or not many other fishes of differentkinds would have a gene homologous to the obtained Pagrus majortransglutaminase cDNA

We investigated as to whether or not a gene which is extremely highlyhomologous to the Pagrus major-derived transglutaminase gene as obtainedby us in the previous example would exist in any other fishes ofdifferent kinds.

As test samples, used were the livers of horse mackerel, youngyellowtail, sardine, Pacific saury, mackerel, bonito and salmon and theovary of globefish; and as a negative control sample, used was thechromosome of Bacillus subtilis. About 3 g of the tissues of therespective fishes were cut into fine pieces, and 30 ml of an ice-cooledTN buffer (20 mM tris-HCl buffer (pH 7.5) containing 0.1M sodiumchloride, as its composition) was added thereto and crushed with ateflon homogenizer.

The sample suspension was put in a centrifugal tube and subjected tocentrifugation with a cooling centrifuger at 5° C. and at 1,500 rpm for5 minutes to obtain a precipitated fraction. To this was added 5 ml ofan ice-cooled TNE buffer (TN buffer containing 1 mM EDTA, as itscomposition) and well suspended. Then, 15 ml of the ice-cooled TNEbuffer was further added thereto and mixed. Afterwards, 1 ml of 10% SDSwas added thereto and shaken at room temperature for 30 minutes, then100 microliter of 20 mg/ml Protease K solution was added thereto andreacted overnight at 50° C.

After the reaction, the aqueous solution of each sample was subjected tophenol treatment, phenol-chloroform treatment and chloroform treatmentfor removal of proteins therefrom. Afterwards, 1/50 volume of 5M sodiumchloride and 2.5 times volume of ethanol were added to each sample so asto precipitate the nucleic acid. This was recovered and finallydissolved in 1 ml of a TE buffer (10 mM tris-HCl buffer (pH 7.6)containing 1 mM EDTA, as its composition).

Next, about 10 microgram of the DNA of each sample was digested with 200units of restriction enzyme HindIII and subjected to electrophoresisusing 1% agarose gel. The gel was stained with ethidium bromide toascertain that the amount of the migrated DNA of each sample was almostconstant. Then, the gel was dipped in 0.25N hydrochloric acid andsubsequently treated with a 0.4N sodium hydroxide solution containing0.6M sodium chloride. Next, the gel was neutralized with a 0.5M tris-HClbuffer (pH 7.5) containing 1.5M sodium chloride, and the nucleic acid inthe gel was transcribed to a nylon membrane (Gene Screen Plus) with 10×SCC (composition: 1.5M sodium chloride, 0.15M trisodium citrate).

The membrane with the nucleic acid as adsorbed thereto was treated with0.4N sodium hydroxide containing 0.6M sodium chloride, then with a 0.5Mtris-HCl buffer (pH 7.5) containing 1.5M sodium chloride, and thereafterdipped in a 2× SCC solution. Afterwards, the membrane was left at roomtemperature for 30 minutes and then dried at 80° C. for 2 hours.

The thus obtained nylon membrane was subjected to prehybridization at65° C. for 3 hours and then to hybridization at 60° C. for 16 hours. Thecomposition of the prehybridization solution was 5× SSC (composition of1× SCC: 0.15M NaCl, 0.015M sodium citrate, pH 7.0), 1× Denhardt'ssolution (composition of 1× Denhardt's solution: 0.02% BSA, 0.02%Ficoll, 0.02% polyvinyl pyrrolidone) and 0.1% SDS. The composition ofthe hybridization solution comprised 0.75M sodium chloride, 20 mMtris-HCl (pH 8.0), 0.25 mM EDTA, 1% SDS, 1× Denhardt's solution and 50microgram/ml of E. coli genome solution. The DNA probe as prepared inthe manner mentioned below was used in the form of having aconcentration of 2×10⁵ cpm/ml.

As the DNA probe, used was a DNA fragment as radio isotope-labeled witha random primer, using guinea pig transglutaminase cDNA fragment and thePagrus major transglutaminase cDNA fragment as obtained by us each as atemplate. As the guinea pig transglutaminase cDNA fragment, used was onein which the plasmid pKTG1 (Ikura et al, Eur. J. Biochem., Vol. 187, pp.705-711, (1990)) had been treated with restriction enzyme StuI. For thePagrus major transglutaminase cDNA, the obtained cDNA clone pSLTG5 wascleaved with restriction enzymes ApaI and SacI, and the resultingtransglutaminase cDNA fragment was used as a template.

The membrane which had been subjected to the hybridization mentionedabove was washed with 0.1× SCC and 0 1% SDS solution at 60° C. dried andthen subjected to autoradiography. As a result, it was found that theguinea pig-derived DNA probe did not hybridize to the nucleic acid ofall the test samples under the condition of this experiment; while thePagrus major-derived DNA probe did not hybridize to the chromosomal DNAof B. subtilis at all but strongly hybridized to the nucleic acidsderived from horse mackerel, bonito, mackerel, young yellowtail andglobefish and to, though weakly, those derived from sardine, Pacificsaury and salmon.

From the above-mentioned facts, the existence of a gene having a highhomology to the transglutaminase gene as obtained by us in other fishesof different kinds was verified for the first time; and it was alsoverified for the first time that a transglutaminase gene may also beobtained with extreme ease even from other fishes of different kindsthan Pagrus major, Alaska pollack, Theragra chalogramma, and flounder,Paralichthys olivaceus, by the use of the transglutaminase as obtainedby us as a DNA probe.

8. Analysis of partial amino acid sequence of Alaska pollack livertransglutaminase

In order to show the fact that the Alaska pollack-derivedtransglutaminase gene as obtained in the manner mentioned above is oneto actually express a transglutaminase enzyme in an Alaska pollack, thenatural enzyme was purified and the structure thereof was clarified. Thetransglutaminase activity of the enzyme was verified according to anactivity detection method which measures the change in fluorescenceintensity due to bonding of monodansylcadaverine with dimethylatedcasein as an index, in accordance with the activity detecting methodmentioned above.

30 ml of a 20 mM tris-HCl buffer (pH 8.3) containing 10 mM NaCl, 5 mMEDTA and 2 mM dithiothreitol was added to 15 g of Alaska pollack liverand crushed with a homogenizer. The thus-obtained suspension wascentrifuged at 4° C., 3,000 rpm for 10 minutes (Hitachi's Himac CR 20B2,with rotor RPR20-2). Then, the supernatant was further centrifuged at 4°C., at 37,000 rpm for one hour (Hitachi's 70P-72, with rotor RP-70T).The supernatant was filtered through a 0.45 micrometer-filter (GLScience's GL Chromatodisc) to obtain 24 ml of a crude extract.

The protein concentration of the extract was measured with a BioRad'sprotein assay kit to be about 8.6 mg/ml. Using 5 microliter of the crudeextract, the transglutaminase activity thereof was determined to have atotal activity of 849 units. Thus, the relative activity of it was 4.10units/mg protein.

Next, the crude extract was passed through a Q-Sepharose column(Pharmacia; diameter 1.6 cm×10 cm) as equilibrated with the same buffer,whereupon the ion exchanger of the column adsorbed the transglutaminase.Then, NaCl concentration gradient elution of the column was effected togive a fraction having a transglutaminase activity at the NaClconcentration of about 100 mM. The thus obtained transglutaminase-activefraction (10 ml) was subjected to dialysis to the same buffer overnight,again passed through the Q-Sepharose column and subjected to elutionunder the same condition to obtain 9.5 ml of a fraction having atransglutaminase-activity.

The protein concentration of the active fraction was about 0.73 mg/ml.The total transglutaminase activity of it was 249 units, and thereforethe relative activity of it was 36.1 units/mg protein.

Next, the thus obtained active fraction was subjected to dialysisovernight to a sodium acetate solution (pH 6.45) containing 50 mm NaCl,2 mM EDTA and 0.5 mM dithiothreitol and applied to through anS-Sepharose column (Pharmacia: diameter 1.6 cm×10 cm) as equilibratedwith the same buffer, whereupon the column adsorbed thetransglutaminase. Then, NaCl concentration gradient elution of thecolumn was effected to give a fraction (6.0 ml) having atransglutaminase activity at the NaCl concentration of about 200 mM.

The protein concentration of the active fraction was about 56microgram/ml. The total transglutaminase activity of it was 201 units,and therefore the relative activity of it was 591.2 units/mg protein.

In the fraction, only a protein showing a single band of a molecularweight of about 77,000 on SDS-PAGE exited, and thus an Alaskapollack-derived transglutaminase was purified and obtained. The relativeactivity of the purified fraction was 143 times as large as that of thecrude extract; and the recovery yield of the former was 23.7%.

(a) Electrophoresis Assay

30 microliters of a purified transglutaminase solution was added withthe same amount of a 0.125M tris-HCl buffer (pH 6.8) containing 10%mercaptoethanol, 4% SDS, 20% glycerin and 0.002% bromophenol blue, andheated in a boiling bath for one minute to prepare a sample forelectrophoresis. 40 microliters of the sample was applied to a prepared5 to 20% polyacrylamide gel (ATTO) and subjected to electrophoresis witha 0.025M tris-glycine buffer containing 0.1% SDS for about 2 hours at 40mA. After completion of the process, the migrated sample was stainedovernight with a 0.12% Coomassie brilliant blue solution containing 50%methanol and 7% acetic acid and then decolored with a 7% acetic acidsolution containing 50% methanol. As a result, a single band wasobtained at a molecular weight of about 77,000.

(b) Partial amino acid sequence of Alaska pollack liver-derivedtransglutaminase

About 4 ml of the S-Sepharose fraction containing about 80 microgram ofthe purified transglutaminase was put in a dialytic tube and subjectedto dialysis to a 5 mM tris-HCl buffer (pH 8.3) containing 0.001 mm EDTAfor 6 hours, then again to dialysis to the same solution, whereby thealkali metal ions were removed from the transglutaminase product. Thiswas dried by centrifugal concentration, 0.8 ml of distilled water wasadded thereto and stirred at 37° C. for 30 minutes so that the productwas again dissolved in the water. To this was added 16 microgram oftrypsin (by Sigma; 11,700 units/mg) and reacted at 37° C. for 12 hours,whereby this was fragmented into peptide fragments. The reaction wasterminated by adding one drop of formic acid thereto.

Next, the reaction liquid was applied to a reverse phase HPLC (InertsilPrep-ODS; diameter 6.0 mm×250 mm; by GL Science), using a solvent of0.05% TFA (trifluoroacetic acid), whereupon the respective peptidefragments were separated and collected by acetonitrile concentrationgradient elution.

The thus obtained peptide fragments were applied to a protein sequencer(MilliGen Biosearch; 6400/6600) to determine their amino acid sequences.The following sequences were obtained:

    ______________________________________    Xaa--Ala--Gly--Gly--Ser--Gly--Asp (SEQ ID NO:46);    Trp--Trp--Leu--His--Gln--Gln--Ser (SEQ ID NO:47);    Met--Tyr--Leu--Leu--Phe--Asn--Pro (SEQ ID NO:48);    Trp--Gln--Glu--Pro--Tyr--Thr--Gly--Gly (SEQ ID NO:49);    Phe--Asp--Val--Pro--Phe--Val--Phe--Ala--Glu--Val--    Asn--Ala--Asp (SEQ ID NO:50); and    Ser--Xaa--Tyr--Ser--Asn--Glu (SEQ ID NO:51).    ______________________________________

Xaa indicates an unidentified amino acid residue.

The above-mentioned six amino acid sequences completely correspond to apart of the amino acid sequences (SEQ ID NOS:8 and 10) presumed from thecorresponding base sequence of the cDNA already obtained by us. The factindicated that the cDNA obtained by us was just one which had beenexpressed in the body of Alaska pollack and which coded for the activetransglutaminase.

9. Analysis of partial amino acid sequence of Pagrus major livertransglutaminase

On the other hand, in order to show the fact that the Pagrusmajor-derived transglutaminase gene as obtained in the manner mentionedabove is one to actually express a transglutaminase enzyme in a pagrusmajor, the natural enzyme was purified and the structure thereof wasclarified. Like the case of the previous Alaska pollack-derived enzyme,the transglutaminase activity of the enzyme of the present case wasverified according to an activity detection method which measures thechange in fluorescence intensity due to bonding of monodansylcadaverinewith dimethylated casein as an index, in accordance with the activitydetecting method mentioned above.

46 ml of a 20 mM Tris-HCl buffer (pH 8.3) containing 10 mM NaCl, 5 mMEDTA and 2 mM dithiothreitol was added to 20 g of Pagrus major liver andcrushed with a homogenizer. The thus obtained suspension was put in acentrifugal tube and applied to a centrifuge (Hitachi's 70P-72, withrotor RPRP-65T) at 4° C., 50,000 rpm for 45 minutes. Then, thesupernatant part was passed through a 0.45 micrometer-filter (AdvanticDISMIC-25 disposable syringe filter unit) so that insoluble high polymersubstances were removed. As a result, 30 ml of an extremely red-coloredextract was obtained. Next, almost the same amount (30 ml) of anice-cooled 5 mM Tris-HCl buffer (pH 8.3) was added to the extract sothat the ionic strength of the resulting solution was lowered. This wasa crude extract of Pagrus major liver (60 ml).

The protein concentration of the extract was measured with a BioRadprotein assay kit to be about 8.4 mg/ml. Using 5 micrometers of thecrude extract, the transglutaminase activity thereof was determined tohave a total activity of 2088 units. Thus, the relative activity of itwas 4.14 units/mg protein.

Next, the crude extract was passed through a DEAE-Sephacel column(Pharmacia; diameter 2.6 cm×11 cm) as equilibrated with a 10 mM Tris-HClbuffer (pH 8.3) containing 5 mM NaCl, 2.5 mM EDTA and 0.5 mMdithiothreitol, whereupon the ion exchanger of the column was found toadsorb the transglutaminase. NaCl concentration gradient elution of thecolumn was effected to give a fraction having a transglutaminaseactivity at the NaCl concentration of about 100 mM. The fraction was aDEAE fraction (about 59 ml).

The protein concentration of the active fraction was about 145microgram/ml. The total transglutaminase activity of it was 1045 units,and therefore the relative activity of it was 122 units/mg protein.

The transglutaminase-active fraction thus fractionated with theDEAE-Sephacel resin was put in a dialytic tube and subjected to dialysisovernight to a 20 mM sodium acetate buffer (pH 6.25) containing 2 mMEDTA and 0.5 mM dithiothreitol. Next, the product was passed through aCM-Sepharose column (Pharmacia; diameter 1.6 cm×10 cm) as equilibratedwith the same buffer, whereupon the column adsorbed thetransglutaminase. Then, NaCl concentration gradient elution of thecolumn was effected to give a transglutaminase-active fraction at theNaCl concentration of about 200 mM. The fraction (about 35 ml) wasobtained to be a CM fraction.

The protein concentration of the active fraction was about 20microgram/ml. The total transglutaminase activity of it was about 530.6units, and therefore the relative activity of it was 758 units/mgprotein.

The transglutaminase-active fraction thus fractionated with theCM-Sepharose resin was again put in a dialytic tube and subjected todialysis overnight to a 20 mM sodium acetate buffer (pH 6.45) containing50 mM NaCl, 2 mM EDTA and 0.5 mM dithiothreitol so that the saltconcentration in the transglutaminase fraction liquid was lowered. Thiswas passed through a heparin-Sepharose column (Pharmacia; Hi-Trapaffinity column; bed-volume 1 ml) as equilibrated with the same buffer,whereupon the resin adsorbed the transglutaminase. NaCl concentrationgradient elution of the column was effected, whereupon thetransglutaminase was eluted from the heparin column at the NaClconcentration of about 200 mM. This was obtained (about 12.5 ml). Thiswas a heparin fraction.

The protein concentration of the active fraction was about 32microgram/ml. The total transglutaminase activity of it was about 290.5units, and therefore the relative activity of it was 807 units/mgprotein.

The present heparin fraction was subjected to SDS-polyacrylamide gelelectrophoresis, and then the gel was stained with Coomassie brilliantblue, whereupon a single band as stained at only the position of amolecular weight of about 77,000 was identified. Thus, a Pagrus majorliver-derived transglutaminase was purified and obtained. The relativeactivity of the thus obtained pure transglutaminase fraction was about195 times as high as that of the crude extract and the recovery yield ofthe former was about 14%.

The partial amino acid sequence of the Pagrus major liver-derivedtransglutaminase was analyzed. About 4 ml of the heparin fractioncontaining about 100 micrograms of the purified transglutaminase was putin a dialytic tube and subjected to dialysis for 13 hours to a 20 mMTris-HCl buffer (pH 8.3) containing 0.1 mM EDTA and 0.01 mMdithiothreitol, and then to a 20 mM Tris-HCl buffer (pH 8.3) containing0.001 mM EDTA, whereby the alkali metal ions in the transglutaminaseenzyme product were removed. To this was added 480 mg of urea, and themixture was treated at 37° C. for 30 minutes. Then, 7.5 micrograms (0.02unit amidase activity) of lysyl endo-peptidase (Wako Pure Chemicals) wasadded thereto and enzymatically treated therewith at 37° C. for 12hours, so that the transglutaminase was fragmented to peptide fragments.After the treatment, 40 microliters of a 10% TFA (trifluoroacetic acid)solution was added to the reaction liquid and stirred, the finalconcentration of TFA being 0.1%.

Next, the reaction liquid was applied to a reversed phase HPLC (Vydac'sC4 column; diameter 4.6 mm×250 mm), using a solvent of 0.1% TFA, foracetonitrile concentration gradient elution to separate the respectivefragments, which were recovered.

The thus obtained peptide fragments were applied to a protein sequencer(Applied Biosystems; 470A) and the amino acid sequence of each of themwas analyzed with a sequence analyzer (Applied Biosystems; 120A). Theresulting sequences are identified as SEQ ID NOS:52-68 (See theaccompanying Sequence Listing). Xaa indicates an unidentified amino acidresidue.

These amino acid sequences are in the amino acid sequence (SEQ ID NOS:4and 6) to be derived from the cDNA sequence obtained in the previousExample 1. The fact surely indicates that the cDNA as obtained hereincodes for the enzyme showing the transglutaminase activity asfunctioning in a living body of a Pagrus major.

10. Comparison of the enzymatic characteristics between guineapig-derived transglutaminase add fish-derived transglutaminase

The enzymatic characteristics of fish-derived transglutaminase(hereinafter referred to as FTG) were compared with those of guinea pig(marmot)-derived transglutaminase (hereinafter referred to as MTG), andthe differences between them were investigated. In particular, theindustrial superiority of FTG to MTG was investigated by the comparison.

(a) Relative activity

The enzymatic relative activity of the pure Alaska pollacktransglutaminase as obtained in the previous example was compared withthat of a commercial MTG by a fluorescent method. MTG samples usedherein were one from Takara Shuzo and one from Sigma. Takara Shuzo's MTGhad a purity of 95%; and Sigma's MTG had a similar purity.

The above-mentioned three transglutaminase products were prepared tohave the same enzymatic concentration (as measured by Biorad's proteinassay kit for the protein concentration), and the transglutaminaseactivity of each sample was measured at a pH value of 8.5 of being nearto the respective optimum pH. As a result, it was found that therelative activity of FTG was 10 times as high as that of MTG (TakaraShuzo) and was 20 times as high as that of MTG (Sigma).

The above-mentioned three transglutaminase products each having acontrolled concentration were developed with SDS-PAGE, stained withCoomassie Brilliant Blue and applied to a densitometer (LKB; Ultro ScanXL Laser Densitometer) to determine the quantity of the transglutaminaseof each sample product. As a result, it was found that thetransglutaminase quantity in FTG was about 4 times as high as those ofthe MTG'S. From the fact, it was found that the transglutaminaserelative activity of FTG by a fluorescent method was 2.5 to 5 times ashigh as that of MTG.

As mentioned above, the relative activity of FTG is higher than that ofMTG. Thus, in view of the industrial use of the enzyme, the amount ofthe FTG to be used for attaining expression of the same effect may bereduced below that of the MTG. Accordingly, the industrial advantage ofFTG is noticeable because of the reduction of the cost of producing it.

(b) Thermal stability

Next, FTG and MTG were compared with each other with respect to theeasiness of thermal deactivation of them. The amount of the enzyme usedfor the test was such that would show the activity of the same degree.Each transglutaminase product was first put in a Tris-HCl buffer (pH8.5) (in the form of a solution comprising 250 microliters of 0.5MTris-HCl buffer (pH 8.5), 80 microliters of 100 mM DTT, 37 microlitersof 1 mM monodansylcadaverine and 1300 microliters of water as thecomposition) and treated at 0° C., 20° C., 25° C., 30° C., 37° C., 40°C., 42° C., 50° C. or 60° C. for 10 minutes, then cooled with ice for 3minutes to stop the thermal treatment of the product. Next, 250microliters of 10 mg/ml dimethylated casein solution and 500 microlitersof 50 mM calcium chloride solution were added thereto and reacted at 37°C. for 60 minutes. Afterwards, 100 microliters of 0.5M EDTA was added toeach of the reaction liquids so that the enzymatic reaction was stopped.

The fluorescent intensity of each of the thus prepared reaction liquidswas measured in the manner as mentioned above, whereby the thermalstability of the enzyme tested was determined. As a result, it wasclarified that the temperature of treating FTG for giving the residualactivity of 50% was lower than that of MTG by about 9° C. as shown inFIG. 13. The fact indicates that FTG is more easily deactivated thanMTG. In combination with the result in the previous relative activitystudy (a), it is concluded that FTG has a higher enzymatic reactivityand nay more easily be deactivated by mere heat treatment of thereaction product than MTG.

(c) Reactivity with actomyosin

Actomyosin (AM) was prepared in the manner mentioned below. First, about60 ml of a 20 mM Tris-HCl buffer (pH 7.5) containing 0.5M sodiumchloride was added to about 30 g of frozen surimi of Alaska pollack (SAgrade product by Taiyo Fishery) and homogenized with ExcellautoHomogenizer (manufactured by Nissei Industry). This was then subjectedto centrifugation three times, each time at 10,000 rpm for 30 seconds,using Hitachi's Himac CR20B2 Model Centrifuge (with rotor of RPR20-2),so that insoluble substances were removed therefrom. The resultingsupernatant was put in a dialytic tube (Seamless Cellulose Tubing,imported by Sanko Pure Chemicals) and subjected to dialysis to a 20 mMTris-HCl buffer (pH 7.5) containing 0.5M sodium chloride for 3 hours andthen for 16 hours.

Afterwards, the dialyzed liquid was subjected to centrifugation at14,000 rpm for 60 minutes to obtain the supernatant. This was filteredthrough a cotton gauze cloth to prepare an actomyosin solution (about 27ml). The protein concentration of the solution was measured withBioRad's Protein Assay Kit to be 26.1 mg/ml.

Using the actomyosin as prepared in the manner mentioned above, MTG andFTG were compared with each other with respect to the action and effectthereof on polymerization of the myocin protein. Studies show thatpolymerization of myocin is an index having a close relation to the"good taste" of marine paste products, including the elasticitydisplaying function of kamaboko (boiled fish paste product) (see NipponSuisan Gakkaishi, vol. 51, pp. 1559-1565 (1985) and Nippon SuisanGakkaishi, vol. 56, pp. 125-132 (1990)).

The actomyosin as prepared in the manner mentioned above was formed intoa solution having a concentration of 10 mg/ml, using a 20 mM Tris-HClbuffer (pH 7.5) containing 0.5M sodium chloride, and one ml of this wasput in a test tube (washed test tube Larbo, by Terumo; 15.5×100 mm).Plural test tubes each containing it were prepared. To each of them wasadded 200 microliters of 50 mM calcium chloride and stirred. Then, 200microliters of a separately prepared FTG or MTG (both having the sameactivity as measured by a fluorescent activity measuring method) wasadded thereto and fully stirred. MTG was one produced by Sigma.

After addition of each transglutaminase, it was reacted at 37° C. for 15minutes, 30 minutes, 45 minutes and 60 minutes. After the reaction, 100microliters of the reaction solution was sampled from each test tube,and 100 microliters of a 20 mM Tris-HCl buffer (pH 8.5) containing 8Murea, 2% SDS and 2% beta-mercaptoethanol was added thereto so that thereaction was stopped. Next, 300 microliters of a 125 mM Tris-HCl buffer(pH 6.8) containing 4% SDS, 10% beta-mercaptoethanol, 20% glycerin and0,002% bromophenol blue was added thereto, and the sample was thenheated at 100° C. for one minute to give a sample for SDS-PAGE.

5 microliters of each of the thus prepared samples was applied toSDS-PAGE for electrophoresis, then stained with Coomassie BrilliantBlue, and the monomer amount of the myocin H chain was measured with adensitometer (Ultro Scan XL Laser Densitometer, by LKB). From this, theeffect of the transglutaminase tested on the polymerization of themyocin protein was analyzed. The result is shown in FIG. 14, from whichit was found that FTG could more rapidly polymerize the myocin proteinthan MTG. The fact further indicates that FTG is better than MTG also inapplications of the transglutaminase enzyme to marine paste productsbecause of reduction of the manufacture cost.

In the past, the sources which could be used to supply transglutaminasehave included actinomyces and guinea pigs, but the present inventionprovides a DNA fragment coding for a fish-derived transglutaminase, apolypeptide which retains and reinforces the texture of traditionalprocessed marine products, such as the boiled fish paste known as"kamaboko" etc and the function of which is known in naturally occurringsubstances. By applying recombinant technology to the fishtransglutaminase gene obtained according to the present invention, massproduction of transglutaminase is possible. Further, applications of therecombinantly-produced enzyme may result in altering the properties ofedible protein and improving the nutritional value of foods. In additionto food products, this enzyme may be applied to processing andpreparation of pharmaceuticals, and in the manufacture of chemicalproducts.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that, within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 72    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 125 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    AATTCATCGATTAGTAAGGAGGTTTAAAATGGCTTCTTATAAAGGTCTGATTGTTGATGT60    TAATGGTCGTTCTCATGAAAACAACCTGGCACATCGTACGCGTGAAATCGACCGTGAGCG120    CCTGA125    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 8 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    TyrGlyGlnCysTrpValPheAla    15    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2085 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA to mRNA    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Pagrus major    (F) TISSUE TYPE: liver    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..2082    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    GCCAGCTACAAGGGGTTGATTGTTGATGTGAATGGGAGAAGTCATGAA48    AlaSerTyrLysGlyLeuIleValAspValAsnGlyArgSerHisGlu    151015    AACAACTTGGCTCACCGCACCAGGGAGATTGATCGGGAGCGCCTGATC96    AsnAsnLeuAlaHisArgThrArgGluIleAspArgGluArgLeuIle    202530    GTCCGCAGAGGTCAACCCTTCTCCATCACTTTGCAGTGCTCTGACTCT144    ValArgArgGlyGlnProPheSerIleThrLeuGlnCysSerAspSer    354045    CTGCCGCCCAAACACCACCTGGAGCTGGTCCTGCACCTCGGTAAGAGA192    LeuProProLysHisHisLeuGluLeuValLeuHisLeuGlyLysArg    505560    GACGAGGTGGTGATCAAGGTTCAGAAGGAACATGGGGCCAGAGACAAG240    AspGluValValIleLysValGlnLysGluHisGlyAlaArgAspLys    65707580    TGGTGGTTTAACCAGCAGGGAGCTCAGGATGAAATACTGCTGACTCTG288    TrpTrpPheAsnGlnGlnGlyAlaGlnAspGluIleLeuLeuThrLeu    859095    CACAGCCCAGCGAACGCTGTCATTGGCCACTACCGTCTGGCTGTGTTG336    HisSerProAlaAsnAlaValIleGlyHisTyrArgLeuAlaValLeu    100105110    GTGATGTCACCAGATGGTCACATCGTAGAGAGGGCAGACAAAATTAGC384    ValMetSerProAspGlyHisIleValGluArgAlaAspLysIleSer    115120125    TTCCACATGCTCTTCAACCCGTGGTGCAGAGATGATATGGTTTACCTC432    PheHisMetLeuPheAsnProTrpCysArgAspAspMetValTyrLeu    130135140    CCTGATGAGAGTAAGCTCCAGGAGTATGTCATGAATGAAGATGGAGTG480    ProAspGluSerLysLeuGlnGluTyrValMetAsnGluAspGlyVal    145150155160    ATTTACATGGGGACCTGGGATTACATCAGAAGTATACCCTGGAATTAT528    IleTyrMetGlyThrTrpAspTyrIleArgSerIleProTrpAsnTyr    165170175    GGACAGTTTGAGGACTATGTGATGGACATCTGTTTTGAAGTCTTGGAC576    GlyGlnPheGluAspTyrValMetAspIleCysPheGluValLeuAsp    180185190    AACTCCCCAGCTGCCTTGAAAAACTCAGAGATGGACATTGAGCACAGA624    AsnSerProAlaAlaLeuLysAsnSerGluMetAspIleGluHisArg    195200205    TCAGACCCCGTCTATGTCGGCAGGACAATCACTGCAATGGTGAACTCT672    SerAspProValTyrValGlyArgThrIleThrAlaMetValAsnSer    210215220    AACGGTGACAGGGGTGTGTTGACTGGTCGCTGGGAGGAGCCGTACACT720    AsnGlyAspArgGlyValLeuThrGlyArgTrpGluGluProTyrThr    225230235240    GATGGGGTCGCACCGTATCGATGGACCGGCAGCGTGCCGATCCTCCAA768    AspGlyValAlaProTyrArgTrpThrGlySerValProIleLeuGln    245250255    CAGTGGAGCAAGGCCGGGGTGAGGCCGGTCAAATATGGCCAGTGCTGG816    GlnTrpSerLysAlaGlyValArgProValLysTyrGlyGlnCysTrp    260265270    GTGTTTGCTGCCGTCGCCTGCACAGTGCTGCGCTGCCTGGGAATCCCA864    ValPheAlaAlaValAlaCysThrValLeuArgCysLeuGlyIlePro    275280285    ACACGCCCCATCACCAACTTCGCTTCAGCCCATGATGTCGATGGTAAC912    ThrArgProIleThrAsnPheAlaSerAlaHisAspValAspGlyAsn    290295300    CTCTCGGTAGACTTCCTGCTGAATGAGAGACTGGAGAGCTTGGACAGT960    LeuSerValAspPheLeuLeuAsnGluArgLeuGluSerLeuAspSer    305310315320    AGACAGAGAAGTGACAGTAGCTGGAACTTCCACTGTTGGGTTGAATCC1008    ArgGlnArgSerAspSerSerTrpAsnPheHisCysTrpValGluSer    325330335    TGGATGAGCAGAGAGGATCTCCCTGAAGGAAATGATGGCTGGCAGGTT1056    TrpMetSerArgGluAspLeuProGluGlyAsnAspGlyTrpGlnVal    340345350    TTGGATCCCACCCCTCAAGAACTGAGTGATGGTGAGTTTTGCTGTGGT1104    LeuAspProThrProGlnGluLeuSerAspGlyGluPheCysCysGly    355360365    CCGTGTCCAGTGGCGGCCATCAAGGAGGGAAATCTGGGAGTGAAGTAC1152    ProCysProValAlaAlaIleLysGluGlyAsnLeuGlyValLysTyr    370375380    GACGCCCCCTTTGTATTCGCTGAGGTGAACGCTGACACCATCTACTGG1200    AspAlaProPheValPheAlaGluValAsnAlaAspThrIleTyrTrp    385390395400    ATCGTCCAAAAAGATGGCCAACGACGGAAGATCACAGAGGACCATGCT1248    IleValGlnLysAspGlyGlnArgArgLysIleThrGluAspHisAla    405410415    AGTGTGGGGAAGAACATCAGCACAAAAAGCGTTTACGGCAACCACAGA1296    SerValGlyLysAsnIleSerThrLysSerValTyrGlyAsnHisArg    420425430    GAAGATGTCACTCTGCACTACAAATATCCTGAAGGCTCCCAGAAGGAG1344    GluAspValThrLeuHisTyrLysTyrProGluGlySerGlnLysGlu    435440445    AGGGAAGTGTACAAGAAGGCGGGACGCCGGGTCACAGAGCCATCCAAC1392    ArgGluValTyrLysLysAlaGlyArgArgValThrGluProSerAsn    450455460    GAGATCGCAGAACAAGGAAGACTTCAGCTGTCAATCAAGCATGCCCAG1440    GluIleAlaGluGlnGlyArgLeuGlnLeuSerIleLysHisAlaGln    465470475480    CCTGTATTTGGGACAGACTTTGATGTGATTGTTGAGGTGAAGAATGAA1488    ProValPheGlyThrAspPheAspValIleValGluValLysAsnGlu    485490495    GGAGGCAGAGATGCTCATGCTCAGCTGACCATGCTGGCCATGGCAGTA1536    GlyGlyArgAspAlaHisAlaGlnLeuThrMetLeuAlaMetAlaVal    500505510    ACTTACAATTCTCTCCGCCGGGGGGAGTGCCAGAGAAAAACAATCAGT1584    ThrTyrAsnSerLeuArgArgGlyGluCysGlnArgLysThrIleSer    515520525    GTGACTGTGCCCGCTCACAAAGCCCACAAGGAGGTTATGCGTCTGCAC1632    ValThrValProAlaHisLysAlaHisLysGluValMetArgLeuHis    530535540    TACGACGACTATGTCAGGTGTGTCTCTGAGCATCACCTGATCAGGGTG1680    TyrAspAspTyrValArgCysValSerGluHisHisLeuIleArgVal    545550555560    AAAGCGCTCTTAGACGCTCCAGGGGAGAACGGGCCCATCATGACCGTG1728    LysAlaLeuLeuAspAlaProGlyGluAsnGlyProIleMetThrVal    565570575    GCCAACATCCCACTGAGCACGCCTGAACTCCTTGTACAGGTGCCTGGG1776    AlaAsnIleProLeuSerThrProGluLeuLeuValGlnValProGly    580585590    AAGGCTGTTGTATGGGAACCACTGACAGCCTACGTCTCCTTCACCAAT1824    LysAlaValValTrpGluProLeuThrAlaTyrValSerPheThrAsn    595600605    CCTCTGCCAGTTCCTCTGAAGGGTGGCGTTTTCACTTTGGAGGGTGCT1872    ProLeuProValProLeuLysGlyGlyValPheThrLeuGluGlyAla    610615620    GGCCTGCTGTCTGCCACTCAGATCCATGTTAATGGTGCTGTAGCTCCA1920    GlyLeuLeuSerAlaThrGlnIleHisValAsnGlyAlaValAlaPro    625630635640    AGTGGGAAAGTGTCTGTCAAGCTCTCTTTCTCCCCCATGCGCACCGGG1968    SerGlyLysValSerValLysLeuSerPheSerProMetArgThrGly    645650655    GTGAGGAAGCTCCTGGTGGACTTTGACTCTGACAGACTGAAGGACGTG2016    ValArgLysLeuLeuValAspPheAspSerAspArgLeuLysAspVal    660665670    AAGGGTGTCACCACCGTGGTTGTCCACAAGAAATACAGATCTCTAATT2064    LysGlyValThrThrValValValHisLysLysTyrArgSerLeuIle    675680685    ACTGGACTTCACACAGACTAA2085    ThrGlyLeuHisThrAsp    690    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 694 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    AlaSerTyrLysGlyLeuIleValAspValAsnGlyArgSerHisGlu    151015    AsnAsnLeuAlaHisArgThrArgGluIleAspArgGluArgLeuIle    202530    ValArgArgGlyGlnProPheSerIleThrLeuGlnCysSerAspSer    354045    LeuProProLysHisHisLeuGluLeuValLeuHisLeuGlyLysArg    505560    AspGluValValIleLysValGlnLysGluHisGlyAlaArgAspLys    65707580    TrpTrpPheAsnGlnGlnGlyAlaGlnAspGluIleLeuLeuThrLeu    859095    HisSerProAlaAsnAlaValIleGlyHisTyrArgLeuAlaValLeu    100105110    ValMetSerProAspGlyHisIleValGluArgAlaAspLysIleSer    115120125    PheHisMetLeuPheAsnProTrpCysArgAspAspMetValTyrLeu    130135140    ProAspGluSerLysLeuGlnGluTyrValMetAsnGluAspGlyVal    145150155160    IleTyrMetGlyThrTrpAspTyrIleArgSerIleProTrpAsnTyr    165170175    GlyGlnPheGluAspTyrValMetAspIleCysPheGluValLeuAsp    180185190    AsnSerProAlaAlaLeuLysAsnSerGluMetAspIleGluHisArg    195200205    SerAspProValTyrValGlyArgThrIleThrAlaMetValAsnSer    210215220    AsnGlyAspArgGlyValLeuThrGlyArgTrpGluGluProTyrThr    225230235240    AspGlyValAlaProTyrArgTrpThrGlySerValProIleLeuGln    245250255    GlnTrpSerLysAlaGlyValArgProValLysTyrGlyGlnCysTrp    260265270    ValPheAlaAlaValAlaCysThrValLeuArgCysLeuGlyIlePro    275280285    ThrArgProIleThrAsnPheAlaSerAlaHisAspValAspGlyAsn    290295300    LeuSerValAspPheLeuLeuAsnGluArgLeuGluSerLeuAspSer    305310315320    ArgGlnArgSerAspSerSerTrpAsnPheHisCysTrpValGluSer    325330335    TrpMetSerArgGluAspLeuProGluGlyAsnAspGlyTrpGlnVal    340345350    LeuAspProThrProGlnGluLeuSerAspGlyGluPheCysCysGly    355360365    ProCysProValAlaAlaIleLysGluGlyAsnLeuGlyValLysTyr    370375380    AspAlaProPheValPheAlaGluValAsnAlaAspThrIleTyrTrp    385390395400    IleValGlnLysAspGlyGlnArgArgLysIleThrGluAspHisAla    405410415    SerValGlyLysAsnIleSerThrLysSerValTyrGlyAsnHisArg    420425430    GluAspValThrLeuHisTyrLysTyrProGluGlySerGlnLysGlu    435440445    ArgGluValTyrLysLysAlaGlyArgArgValThrGluProSerAsn    450455460    GluIleAlaGluGlnGlyArgLeuGlnLeuSerIleLysHisAlaGln    465470475480    ProValPheGlyThrAspPheAspValIleValGluValLysAsnGlu    485490495    GlyGlyArgAspAlaHisAlaGlnLeuThrMetLeuAlaMetAlaVal    500505510    ThrTyrAsnSerLeuArgArgGlyGluCysGlnArgLysThrIleSer    515520525    ValThrValProAlaHisLysAlaHisLysGluValMetArgLeuHis    530535540    TyrAspAspTyrValArgCysValSerGluHisHisLeuIleArgVal    545550555560    LysAlaLeuLeuAspAlaProGlyGluAsnGlyProIleMetThrVal    565570575    AlaAsnIleProLeuSerThrProGluLeuLeuValGlnValProGly    580585590    LysAlaValValTrpGluProLeuThrAlaTyrValSerPheThrAsn    595600605    ProLeuProValProLeuLysGlyGlyValPheThrLeuGluGlyAla    610615620    GlyLeuLeuSerAlaThrGlnIleHisValAsnGlyAlaValAlaPro    625630635640    SerGlyLysValSerValLysLeuSerPheSerProMetArgThrGly    645650655    ValArgLysLeuLeuValAspPheAspSerAspArgLeuLysAspVal    660665670    LysGlyValThrThrValValValHisLysLysTyrArgSerLeuIle    675680685    ThrGlyLeuHisThrAsp    690    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2520 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA to mRNA    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Pagrus major    (F) TISSUE TYPE: liver    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 34..2121    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    CTTTAACAGACTTTGATAGGAAGAAGATCTGCGATGGCCAGCTACAAGGGGTTG54    MetAlaSerTyrLysGlyLeu    15    ATTGTTGATGTGAATGGGAGAAGTCATGAAAACAACTTGGCTCACCGC102    IleValAspValAsnGlyArgSerHisGluAsnAsnLeuAlaHisArg    101520    ACCAGGGAGATTGATCGGGAGCGCCTGATCGTCCGCAGAGGTCAACCC150    ThrArgGluIleAspArgGluArgLeuIleValArgArgGlyGlnPro    253035    TTCTCCATCACTTTGCAGTGCTCTGACTCTCTGCCGCCCAAACACCAC198    PheSerIleThrLeuGlnCysSerAspSerLeuProProLysHisHis    40455055    CTGGAGCTGGTCCTGCACCTCGGTAAGAGAGACGAGGTGGTGATCAAG246    LeuGluLeuValLeuHisLeuGlyLysArgAspGluValValIleLys    606570    GTTCAGAAGGAACATGGGGCCAGAGACAAGTGGTGGTTTAACCAGCAG294    ValGlnLysGluHisGlyAlaArgAspLysTrpTrpPheAsnGlnGln    758085    GGAGCTCAGGATGAAATACTGCTGACTCTGCACAGCCCAGCGAACGCT342    GlyAlaGlnAspGluIleLeuLeuThrLeuHisSerProAlaAsnAla    9095100    GTCATTGGCCACTACCGTCTGGCTGTGTTGGTGATGTCACCAGATGGT390    ValIleGlyHisTyrArgLeuAlaValLeuValMetSerProAspGly    105110115    CACATCGTAGAGAGGGCAGACAAAATTAGCTTCCACATGCTCTTCAAC438    HisIleValGluArgAlaAspLysIleSerPheHisMetLeuPheAsn    120125130135    CCGTGGTGCAGAGATGATATGGTTTACCTCCCTGATGAGAGTAAGCTC486    ProTrpCysArgAspAspMetValTyrLeuProAspGluSerLysLeu    140145150    CAGGAGTATGTCATGAATGAAGATGGAGTGATTTACATGGGGACCTGG534    GlnGluTyrValMetAsnGluAspGlyValIleTyrMetGlyThrTrp    155160165    GATTACATCAGAAGTATACCCTGGAATTATGGACAGTTTGAGGACTAT582    AspTyrIleArgSerIleProTrpAsnTyrGlyGlnPheGluAspTyr    170175180    GTGATGGACATCTGTTTTGAAGTCTTGGACAACTCCCCAGCTGCCTTG630    ValMetAspIleCysPheGluValLeuAspAsnSerProAlaAlaLeu    185190195    AAAAACTCAGAGATGGACATTGAGCACAGATCAGACCCCGTCTATGTC678    LysAsnSerGluMetAspIleGluHisArgSerAspProValTyrVal    200205210215    GGCAGGACAATCACTGCAATGGTGAACTCTAACGGTGACAGGGGTGTG726    GlyArgThrIleThrAlaMetValAsnSerAsnGlyAspArgGlyVal    220225230    TTGACTGGTCGCTGGGAGGAGCCGTACACTGATGGGGTCGCACCGTAT774    LeuThrGlyArgTrpGluGluProTyrThrAspGlyValAlaProTyr    235240245    CGATGGACCGGCAGCGTGCCGATCCTCCAACAGTGGAGCAAGGCCGGG822    ArgTrpThrGlySerValProIleLeuGlnGlnTrpSerLysAlaGly    250255260    GTGAGGCCGGTCAAATATGGCCAGTGCTGGGTGTTTGCTGCCGTCGCC870    ValArgProValLysTyrGlyGlnCysTrpValPheAlaAlaValAla    265270275    TGCACAGTGCTGCGCTGCCTGGGAATCCCAACACGCCCCATCACCAAC918    CysThrValLeuArgCysLeuGlyIleProThrArgProIleThrAsn    280285290295    TTCGCTTCAGCCCATGATGTCGATGGTAACCTCTCGGTAGACTTCCTG966    PheAlaSerAlaHisAspValAspGlyAsnLeuSerValAspPheLeu    300305310    CTGAATGAGAGACTGGAGAGCTTGGACAGTAGACAGAGAAGTGACAGT1014    LeuAsnGluArgLeuGluSerLeuAspSerArgGlnArgSerAspSer    315320325    AGCTGGAACTTCCACTGTTGGGTTGAATCCTGGATGAGCAGAGAGGAT1062    SerTrpAsnPheHisCysTrpValGluSerTrpMetSerArgGluAsp    330335340    CTCCCTGAAGGAAATGATGGCTGGCAGGTTTTGGATCCCACCCCTCAA1110    LeuProGluGlyAsnAspGlyTrpGlnValLeuAspProThrProGln    345350355    GAACTGAGTGATGGTGAGTTTTGCTGTGGTCCGTGTCCAGTGGCGGCC1158    GluLeuSerAspGlyGluPheCysCysGlyProCysProValAlaAla    360365370375    ATCAAGGAGGGAAATCTGGGAGTGAAGTACGACGCCCCCTTTGTATTC1206    IleLysGluGlyAsnLeuGlyValLysTyrAspAlaProPheValPhe    380385390    GCTGAGGTGAACGCTGACACCATCTACTGGATCGTCCAAAAAGATGGC1254    AlaGluValAsnAlaAspThrIleTyrTrpIleValGlnLysAspGly    395400405    CAACGACGGAAGATCACAGAGGACCATGCTAGTGTGGGGAAGAACATC1302    GlnArgArgLysIleThrGluAspHisAlaSerValGlyLysAsnIle    410415420    AGCACAAAAAGCGTTTACGGCAACCACAGAGAAGATGTCACTCTGCAC1350    SerThrLysSerValTyrGlyAsnHisArgGluAspValThrLeuHis    425430435    TACAAATATCCTGAAGGCTCCCAGAAGGAGAGGGAAGTGTACAAGAAG1398    TyrLysTyrProGluGlySerGlnLysGluArgGluValTyrLysLys    440445450455    GCGGGACGCCGGGTCACAGAGCCATCCAACGAGATCGCAGAACAAGGA1446    AlaGlyArgArgValThrGluProSerAsnGluIleAlaGluGlnGly    460465470    AGACTTCAGCTGTCAATCAAGCATGCCCAGCCTGTATTTGGGACAGAC1494    ArgLeuGlnLeuSerIleLysHisAlaGlnProValPheGlyThrAsp    475480485    TTTGATGTGATTGTTGAGGTGAAGAATGAAGGAGGCAGAGATGCTCAT1542    PheAspValIleValGluValLysAsnGluGlyGlyArgAspAlaHis    490495500    GCTCAGCTGACCATGCTGGCCATGGCAGTAACTTACAATTCTCTCCGC1590    AlaGlnLeuThrMetLeuAlaMetAlaValThrTyrAsnSerLeuArg    505510515    CGGGGGGAGTGCCAGAGAAAAACAATCAGTGTGACTGTGCCCGCTCAC1638    ArgGlyGluCysGlnArgLysThrIleSerValThrValProAlaHis    520525530535    AAAGCCCACAAGGAGGTTATGCGTCTGCACTACGACGACTATGTCAGG1686    LysAlaHisLysGluValMetArgLeuHisTyrAspAspTyrValArg    540545550    TGTGTCTCTGAGCATCACCTGATCAGGGTGAAAGCGCTCTTAGACGCT1734    CysValSerGluHisHisLeuIleArgValLysAlaLeuLeuAspAla    555560565    CCAGGGGAGAACGGGCCCATCATGACCGTGGCCAACATCCCACTGAGC1782    ProGlyGluAsnGlyProIleMetThrValAlaAsnIleProLeuSer    570575580    ACGCCTGAACTCCTTGTACAGGTGCCTGGGAAGGCTGTTGTATGGGAA1830    ThrProGluLeuLeuValGlnValProGlyLysAlaValValTrpGlu    585590595    CCACTGACAGCCTACGTCTCCTTCACCAATCCTCTGCCAGTTCCTCTG1878    ProLeuThrAlaTyrValSerPheThrAsnProLeuProValProLeu    600605610615    AAGGGTGGCGTTTTCACTTTGGAGGGTGCTGGCCTGCTGTCTGCCACT1926    LysGlyGlyValPheThrLeuGluGlyAlaGlyLeuLeuSerAlaThr    620625630    CAGATCCATGTTAATGGTGCTGTAGCTCCAAGTGGGAAAGTGTCTGTC1974    GlnIleHisValAsnGlyAlaValAlaProSerGlyLysValSerVal    635640645    AAGCTCTCTTTCTCCCCCATGCGCACCGGGGTGAGGAAGCTCCTGGTG2022    LysLeuSerPheSerProMetArgThrGlyValArgLysLeuLeuVal    650655660    GACTTTGACTCTGACAGACTGAAGGACGTGAAGGGTGTCACCACCGTG2070    AspPheAspSerAspArgLeuLysAspValLysGlyValThrThrVal    665670675    GTTGTCCACAAGAAATACAGATCTCTAATTACTGGACTTCACACAGAC2118    ValValHisLysLysTyrArgSerLeuIleThrGlyLeuHisThrAsp    680685690695    TAAAATAGACATATCTTATATTATGTGATTTTGTGACATTTCCTAGATGTGAGGTGGAGG2178    TGATGTATAAGGTAGATGATATCAACCGCTCAGTGTTATAACAGTTTATAATGCAAATAA2238    GTTCCACTTAAATGATACTGTAGCTATGTCCACGAAGAAAATTCTTGACACAGTGTTAGT2298    TTGATTACCTTAAAGCCTTAAAGCCACTGTATGTCAGATGTGAACTTGTCTGGCTTTGCA2358    TTAAAACCTGGCACATGTTGCTCACATGGAAATGCACAGAAGCACAACAGGTGACGGCCT2418    CTAGATGGAAAATATGTGCGTTTTGTTTCTGTTACTCCTCTGTTTTATTGCCAAATTCAA2478    GATGCTTCCTTCTGTCTTCATTCCAAATGACTGCTGGTTTTT2520    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 695 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    MetAlaSerTyrLysGlyLeuIleValAspValAsnGlyArgSerHis    151015    GluAsnAsnLeuAlaHisArgThrArgGluIleAspArgGluArgLeu    202530    IleValArgArgGlyGlnProPheSerIleThrLeuGlnCysSerAsp    354045    SerLeuProProLysHisHisLeuGluLeuValLeuHisLeuGlyLys    505560    ArgAspGluValValIleLysValGlnLysGluHisGlyAlaArgAsp    65707580    LysTrpTrpPheAsnGlnGlnGlyAlaGlnAspGluIleLeuLeuThr    859095    LeuHisSerProAlaAsnAlaValIleGlyHisTyrArgLeuAlaVal    100105110    LeuValMetSerProAspGlyHisIleValGluArgAlaAspLysIle    115120125    SerPheHisMetLeuPheAsnProTrpCysArgAspAspMetValTyr    130135140    LeuProAspGluSerLysLeuGlnGluTyrValMetAsnGluAspGly    145150155160    ValIleTyrMetGlyThrTrpAspTyrIleArgSerIleProTrpAsn    165170175    TyrGlyGlnPheGluAspTyrValMetAspIleCysPheGluValLeu    180185190    AspAsnSerProAlaAlaLeuLysAsnSerGluMetAspIleGluHis    195200205    ArgSerAspProValTyrValGlyArgThrIleThrAlaMetValAsn    210215220    SerAsnGlyAspArgGlyValLeuThrGlyArgTrpGluGluProTyr    225230235240    ThrAspGlyValAlaProTyrArgTrpThrGlySerValProIleLeu    245250255    GlnGlnTrpSerLysAlaGlyValArgProValLysTyrGlyGlnCys    260265270    TrpValPheAlaAlaValAlaCysThrValLeuArgCysLeuGlyIle    275280285    ProThrArgProIleThrAsnPheAlaSerAlaHisAspValAspGly    290295300    AsnLeuSerValAspPheLeuLeuAsnGluArgLeuGluSerLeuAsp    305310315320    SerArgGlnArgSerAspSerSerTrpAsnPheHisCysTrpValGlu    325330335    SerTrpMetSerArgGluAspLeuProGluGlyAsnAspGlyTrpGln    340345350    ValLeuAspProThrProGlnGluLeuSerAspGlyGluPheCysCys    355360365    GlyProCysProValAlaAlaIleLysGluGlyAsnLeuGlyValLys    370375380    TyrAspAlaProPheValPheAlaGluValAsnAlaAspThrIleTyr    385390395400    TrpIleValGlnLysAspGlyGlnArgArgLysIleThrGluAspHis    405410415    AlaSerValGlyLysAsnIleSerThrLysSerValTyrGlyAsnHis    420425430    ArgGluAspValThrLeuHisTyrLysTyrProGluGlySerGlnLys    435440445    GluArgGluValTyrLysLysAlaGlyArgArgValThrGluProSer    450455460    AsnGluIleAlaGluGlnGlyArgLeuGlnLeuSerIleLysHisAla    465470475480    GlnProValPheGlyThrAspPheAspValIleValGluValLysAsn    485490495    GluGlyGlyArgAspAlaHisAlaGlnLeuThrMetLeuAlaMetAla    500505510    ValThrTyrAsnSerLeuArgArgGlyGluCysGlnArgLysThrIle    515520525    SerValThrValProAlaHisLysAlaHisLysGluValMetArgLeu    530535540    HisTyrAspAspTyrValArgCysValSerGluHisHisLeuIleArg    545550555560    ValLysAlaLeuLeuAspAlaProGlyGluAsnGlyProIleMetThr    565570575    ValAlaAsnIleProLeuSerThrProGluLeuLeuValGlnValPro    580585590    GlyLysAlaValValTrpGluProLeuThrAlaTyrValSerPheThr    595600605    AsnProLeuProValProLeuLysGlyGlyValPheThrLeuGluGly    610615620    AlaGlyLeuLeuSerAlaThrGlnIleHisValAsnGlyAlaValAla    625630635640    ProSerGlyLysValSerValLysLeuSerPheSerProMetArgThr    645650655    GlyValArgLysLeuLeuValAspPheAspSerAspArgLeuLysAsp    660665670    ValLysGlyValThrThrValValValHisLysLysTyrArgSerLeu    675680685    IleThrGlyLeuHisThrAsp    690695    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2088 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA to mRNA    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Theragra chalcogramma    (F) TISSUE TYPE: liver    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..2085    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    GCCCACACAAACCGTTTAATTGCTGGTGTTGATCTGAGAAGCCAGGAA48    AlaHisThrAsnArgLeuIleAlaGlyValAspLeuArgSerGlnGlu    151015    AACAACCGGGAACACCGAACTGAGGAGATTGATAGGAAGCGTTTGATT96    AsnAsnArgGluHisArgThrGluGluIleAspArgLysArgLeuIle    202530    GTTCGGCGGGGACAAGCCTTCTCCCTGACGGTGCACCTCTCCGACCCG144    ValArgArgGlyGlnAlaPheSerLeuThrValHisLeuSerAspPro    354045    CTGCAGTCCGGCCATGAGCTGGCCCTGGTCTTAAAGCAGGATAAGAAC192    LeuGlnSerGlyHisGluLeuAlaLeuValLeuLysGlnAspLysAsn    505560    AACGATGATATTGTGATCAGACAGCGAACGGCTGGAGGGTCTGGTGAC240    AsnAspAspIleValIleArgGlnArgThrAlaGlyGlySerGlyAsp    65707580    AAGTGGTGGTTACACCAGCAGAGCGCGAGGAACGAATTACTGCTGACT288    LysTrpTrpLeuHisGlnGlnSerAlaArgAsnGluLeuLeuLeuThr    859095    GTGTACAGTCCTGCCCGTGCTGCCGTTGGCGAGTACCGCTTGGCTGTT336    ValTyrSerProAlaArgAlaAlaValGlyGluTyrArgLeuAlaVal    100105110    GAACTGATGTCAGGGAATAAACTTCTGGAGAGGACGGACTTTACCAAA384    GluLeuMetSerGlyAsnLysLeuLeuGluArgThrAspPheThrLys    115120125    ATGTACTTGCTGTTTAATCCCTGGTGCAAAGATGATGCTGTGTACCTC432    MetTyrLeuLeuPheAsnProTrpCysLysAspAspAlaValTyrLeu    130135140    CCTGATGAAAGTCTGCTCAAGGAATACATTATGAACGAGAATGGTCGC480    ProAspGluSerLeuLeuLysGluTyrIleMetAsnGluAsnGlyArg    145150155160    ATTTTCACTGGGAGTGCGGATTGGATGAGTGGGTTGCCATGGAATTTC528    IlePheThrGlySerAlaAspTrpMetSerGlyLeuProTrpAsnPhe    165170175    GGACAGTTTGAAGACAATGTGATGGACATCTGCTTTGAGATCCTTGAC576    GlyGlnPheGluAspAsnValMetAspIleCysPheGluIleLeuAsp    180185190    CGCTTTAAGCCAGCAAGGTCAGACCCCCCAAACGACATGCGTCAGCGA624    ArgPheLysProAlaArgSerAspProProAsnAspMetArgGlnArg    195200205    TGGGACCCTGTCTACATCAGCAGGGCAGTCGTTGCCATGGTGAATGCC672    TrpAspProValTyrIleSerArgAlaValValAlaMetValAsnAla    210215220    AACGATGACGGTGGAGTCTTGGTGGGGAAATGGCAGGAACCTTACACA720    AsnAspAspGlyGlyValLeuValGlyLysTrpGlnGluProTyrThr    225230235240    GGTGGAGTACAGCCAACCAAATGGATGAGCAGTGTGCCCATCCTGGAG768    GlyGlyValGlnProThrLysTrpMetSerSerValProIleLeuGlu    245250255    AAGTGGAGCAAATCAAAGTCTGGAGTGAAGTATGGCCAATGCTGGGTG816    LysTrpSerLysSerLysSerGlyValLysTyrGlyGlnCysTrpVal    260265270    TTTGCAGCCGTGGCCTGCACAGTGCTGCGATGCCTGGGCATCCCCACA864    PheAlaAlaValAlaCysThrValLeuArgCysLeuGlyIleProThr    275280285    CGCTGCATCACCAACTTTGAGTCAGCCCATGACACAGACGGAAACCTC912    ArgCysIleThrAsnPheGluSerAlaHisAspThrAspGlyAsnLeu    290295300    TCCATCGACCGAGTGTACAACACACATAGGCAGAGTGTTAACCATGCT960    SerIleAspArgValTyrAsnThrHisArgGlnSerValAsnHisAla    305310315320    GACAGCATCTGGAACTTTCATTGTTGGATCGAGTCTTACATGCAGAGA1008    AspSerIleTrpAsnPheHisCysTrpIleGluSerTyrMetGlnArg    325330335    GAAGATCTACCTGAAGGATATGGTGGCTGGCAAGTCTTGGACCCCACA1056    GluAspLeuProGluGlyTyrGlyGlyTrpGlnValLeuAspProThr    340345350    CCTCAGGAGAGGAGTAGTGGTATGTTTCGCTGTGGCCCATGTCCATTG1104    ProGlnGluArgSerSerGlyMetPheArgCysGlyProCysProLeu    355360365    AAGGCCATTAAAGAAGGGGACCTCAATGTGAAGTTTGATGTTCCATTT1152    LysAlaIleLysGluGlyAspLeuAsnValLysPheAspValProPhe    370375380    GTCTTTGCTGAGGTGAATGCAGACATCATCAATTGGGAAATCAGACCA1200    ValPheAlaGluValAsnAlaAspIleIleAsnTrpGluIleArgPro    385390395400    GACGGTCAGCGAATGCGGCTTTCATCCAACTCCGCAAAAGTGGGGAGG1248    AspGlyGlnArgMetArgLeuSerSerAsnSerAlaLysValGlyArg    405410415    AACATTAGCACCAAAAGTCCTTACAGTAACGAGAGGGAAGATATAACC1296    AsnIleSerThrLysSerProTyrSerAsnGluArgGluAspIleThr    420425430    CTTCAGTACAAGTACCAAGAAGGTTCAGCCAAGGAGCGGGAGGTGTAC1344    LeuGlnTyrLysTyrGlnGluGlySerAlaLysGluArgGluValTyr    435440445    AACAAGGCAGGGCGGCGCATCTCCGGGCCGGATAGAGAAGAGGAATCA1392    AsnLysAlaGlyArgArgIleSerGlyProAspArgGluGluGluSer    450455460    AAACCAGCCAATGAACCAGGAAACGTGCAGCTGGAGATCAGATACGCC1440    LysProAlaAsnGluProGlyAsnValGlnLeuGluIleArgTyrAla    465470475480    AAGCCTGTGTTCGGGACCGACTTTGACGTCATCTTTGAGTTGGAGAAC1488    LysProValPheGlyThrAspPheAspValIlePheGluLeuGluAsn    485490495    ATGGGAGACGAAGAAGTCAGCTGCAAATTGAACATGATGTCAAAGGCT1536    MetGlyAspGluGluValSerCysLysLeuAsnMetMetSerLysAla    500505510    GTCACGTATAACTCGGTCCACCTGGGAGAGTGCCAGAATAGCACAGTC1584    ValThrTyrAsnSerValHisLeuGlyGluCysGlnAsnSerThrVal    515520525    AATGTTGTCATTCCTGCTCACAAAGTCCACAGGGAGACGGTGCGTCTA1632    AsnValValIleProAlaHisLysValHisArgGluThrValArgLeu    530535540    CTCTACACTAAGTATGCATCGTGCGTCAGCGAACACAACATCATCCGG1680    LeuTyrThrLysTyrAlaSerCysValSerGluHisAsnIleIleArg    545550555560    GTGGTAGGGGTGGCAAGAGTGTCCGGCCAGGAAAAATCCATCCTGGAG1728    ValValGlyValAlaArgValSerGlyGlnGluLysSerIleLeuGlu    565570575    ATGGTCAACATCCCACTGAGCAAGCCCAAACTCAGTATTAAGGTTCCT1776    MetValAsnIleProLeuSerLysProLysLeuSerIleLysValPro    580585590    GGCTGGGTGATTTTAAATAGGAAAATCACCACCGTCATCACCTTCACC1824    GlyTrpValIleLeuAsnArgLysIleThrThrValIleThrPheThr    595600605    AATCCATTGCCAGTGCCACTGAACCGAGGAGTGTTCACTGTTGAAGGG1872    AsnProLeuProValProLeuAsnArgGlyValPheThrValGluGly    610615620    GCTGGCCTACTTTCAACCAAAGAGATCCGCATTTCTGGTAGCATCGCT1920    AlaGlyLeuLeuSerThrLysGluIleArgIleSerGlySerIleAla    625630635640    CCAGGCCAGCGTGTGTCTGTGGAGCTGTCCTTCACACCCATGAGGGCG1968    ProGlyGlnArgValSerValGluLeuSerPheThrProMetArgAla    645650655    GGGGTCAGGGAGTTCCTGGTGGACTTTGACTCCGACAGGCTCCAGGAC2016    GlyValArgGluPheLeuValAspPheAspSerAspArgLeuGlnAsp    660665670    GTGAAGGGAGTCGCCACACTGGTGGTCCGCAAGACTTCACCCTCCTAT2064    ValLysGlyValAlaThrLeuValValArgLysThrSerProSerTyr    675680685    TTTCCCATGCCCTACACGTTGTGA2088    PheProMetProTyrThrLeu    690695    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 695 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    AlaHisThrAsnArgLeuIleAlaGlyValAspLeuArgSerGlnGlu    151015    AsnAsnArgGluHisArgThrGluGluIleAspArgLysArgLeuIle    202530    ValArgArgGlyGlnAlaPheSerLeuThrValHisLeuSerAspPro    354045    LeuGlnSerGlyHisGluLeuAlaLeuValLeuLysGlnAspLysAsn    505560    AsnAspAspIleValIleArgGlnArgThrAlaGlyGlySerGlyAsp    65707580    LysTrpTrpLeuHisGlnGlnSerAlaArgAsnGluLeuLeuLeuThr    859095    ValTyrSerProAlaArgAlaAlaValGlyGluTyrArgLeuAlaVal    100105110    GluLeuMetSerGlyAsnLysLeuLeuGluArgThrAspPheThrLys    115120125    MetTyrLeuLeuPheAsnProTrpCysLysAspAspAlaValTyrLeu    130135140    ProAspGluSerLeuLeuLysGluTyrIleMetAsnGluAsnGlyArg    145150155160    IlePheThrGlySerAlaAspTrpMetSerGlyLeuProTrpAsnPhe    165170175    GlyGlnPheGluAspAsnValMetAspIleCysPheGluIleLeuAsp    180185190    ArgPheLysProAlaArgSerAspProProAsnAspMetArgGlnArg    195200205    TrpAspProValTyrIleSerArgAlaValValAlaMetValAsnAla    210215220    AsnAspAspGlyGlyValLeuValGlyLysTrpGlnGluProTyrThr    225230235240    GlyGlyValGlnProThrLysTrpMetSerSerValProIleLeuGlu    245250255    LysTrpSerLysSerLysSerGlyValLysTyrGlyGlnCysTrpVal    260265270    PheAlaAlaValAlaCysThrValLeuArgCysLeuGlyIleProThr    275280285    ArgCysIleThrAsnPheGluSerAlaHisAspThrAspGlyAsnLeu    290295300    SerIleAspArgValTyrAsnThrHisArgGlnSerValAsnHisAla    305310315320    AspSerIleTrpAsnPheHisCysTrpIleGluSerTyrMetGlnArg    325330335    GluAspLeuProGluGlyTyrGlyGlyTrpGlnValLeuAspProThr    340345350    ProGlnGluArgSerSerGlyMetPheArgCysGlyProCysProLeu    355360365    LysAlaIleLysGluGlyAspLeuAsnValLysPheAspValProPhe    370375380    ValPheAlaGluValAsnAlaAspIleIleAsnTrpGluIleArgPro    385390395400    AspGlyGlnArgMetArgLeuSerSerAsnSerAlaLysValGlyArg    405410415    AsnIleSerThrLysSerProTyrSerAsnGluArgGluAspIleThr    420425430    LeuGlnTyrLysTyrGlnGluGlySerAlaLysGluArgGluValTyr    435440445    AsnLysAlaGlyArgArgIleSerGlyProAspArgGluGluGluSer    450455460    LysProAlaAsnGluProGlyAsnValGlnLeuGluIleArgTyrAla    465470475480    LysProValPheGlyThrAspPheAspValIlePheGluLeuGluAsn    485490495    MetGlyAspGluGluValSerCysLysLeuAsnMetMetSerLysAla    500505510    ValThrTyrAsnSerValHisLeuGlyGluCysGlnAsnSerThrVal    515520525    AsnValValIleProAlaHisLysValHisArgGluThrValArgLeu    530535540    LeuTyrThrLysTyrAlaSerCysValSerGluHisAsnIleIleArg    545550555560    ValValGlyValAlaArgValSerGlyGlnGluLysSerIleLeuGlu    565570575    MetValAsnIleProLeuSerLysProLysLeuSerIleLysValPro    580585590    GlyTrpValIleLeuAsnArgLysIleThrThrValIleThrPheThr    595600605    AsnProLeuProValProLeuAsnArgGlyValPheThrValGluGly    610615620    AlaGlyLeuLeuSerThrLysGluIleArgIleSerGlySerIleAla    625630635640    ProGlyGlnArgValSerValGluLeuSerPheThrProMetArgAla    645650655    GlyValArgGluPheLeuValAspPheAspSerAspArgLeuGlnAsp    660665670    ValLysGlyValAlaThrLeuValValArgLysThrSerProSerTyr    675680685    PheProMetProTyrThrLeu    690695    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2921 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA to mRNA    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Theragra chalcogramma    (F) TISSUE TYPE: liver    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 32..2122    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    AGCAACTCTTGGAAAGAATTTAGCAAAGATAATGGCCCACACAAACCGTTTA52    MetAlaHisThrAsnArgLeu    15    ATTGCTGGTGTTGATCTGAGAAGCCAGGAAAACAACCGGGAACACCGA100    IleAlaGlyValAspLeuArgSerGlnGluAsnAsnArgGluHisArg    101520    ACTGAGGAGATTGATAGGAAGCGTTTGATTGTTCGGCGGGGACAAGCC148    ThrGluGluIleAspArgLysArgLeuIleValArgArgGlyGlnAla    253035    TTCTCCCTGACGGTGCACCTCTCCGACCCGCTGCAGTCCGGCCATGAG196    PheSerLeuThrValHisLeuSerAspProLeuGlnSerGlyHisGlu    40455055    CTGGCCCTGGTCTTAAAGCAGGATAAGAACAACGATGATATTGTGATC244    LeuAlaLeuValLeuLysGlnAspLysAsnAsnAspAspIleValIle    606570    AGACAGCGAACGGCTGGAGGGTCTGGTGACAAGTGGTGGTTACACCAG292    ArgGlnArgThrAlaGlyGlySerGlyAspLysTrpTrpLeuHisGln    758085    CAGAGCGCGAGGAACGAATTACTGCTGACTGTGTACAGTCCTGCCCGT340    GlnSerAlaArgAsnGluLeuLeuLeuThrValTyrSerProAlaArg    9095100    GCTGCCGTTGGCGAGTACCGCTTGGCTGTTGAACTGATGTCAGGGAAT388    AlaAlaValGlyGluTyrArgLeuAlaValGluLeuMetSerGlyAsn    105110115    AAACTTCTGGAGAGGACGGACTTTACCAAAATGTACTTGCTGTTTAAT436    LysLeuLeuGluArgThrAspPheThrLysMetTyrLeuLeuPheAsn    120125130135    CCCTGGTGCAAAGATGATGCTGTGTACCTCCCTGATGAAAGTCTGCTC484    ProTrpCysLysAspAspAlaValTyrLeuProAspGluSerLeuLeu    140145150    AAGGAATACATTATGAACGAGAATGGTCGCATTTTCACTGGGAGTGCG532    LysGluTyrIleMetAsnGluAsnGlyArgIlePheThrGlySerAla    155160165    GATTGGATGAGTGGGTTGCCATGGAATTTCGGACAGTTTGAAGACAAT580    AspTrpMetSerGlyLeuProTrpAsnPheGlyGlnPheGluAspAsn    170175180    GTGATGGACATCTGCTTTGAGATCCTTGACCGCTTTAAGCCAGCAAGG628    ValMetAspIleCysPheGluIleLeuAspArgPheLysProAlaArg    185190195    TCAGACCCCCCAAACGACATGCGTCAGCGATGGGACCCTGTCTACATC676    SerAspProProAsnAspMetArgGlnArgTrpAspProValTyrIle    200205210215    AGCAGGGCAGTCGTTGCCATGGTGAATGCCAACGATGACGGTGGAGTC724    SerArgAlaValValAlaMetValAsnAlaAsnAspAspGlyGlyVal    220225230    TTGGTGGGGAAATGGCAGGAACCTTACACAGGTGGAGTACAGCCAACC772    LeuValGlyLysTrpGlnGluProTyrThrGlyGlyValGlnProThr    235240245    AAATGGATGAGCAGTGTGCCCATCCTGGAGAAGTGGAGCAAATCAAAG820    LysTrpMetSerSerValProIleLeuGluLysTrpSerLysSerLys    250255260    TCTGGAGTGAAGTATGGCCAATGCTGGGTGTTTGCAGCCGTGGCCTGC868    SerGlyValLysTyrGlyGlnCysTrpValPheAlaAlaValAlaCys    265270275    ACAGTGCTGCGATGCCTGGGCATCCCCACACGCTGCATCACCAACTTT916    ThrValLeuArgCysLeuGlyIleProThrArgCysIleThrAsnPhe    280285290295    GAGTCAGCCCATGACACAGACGGAAACCTCTCCATCGACCGAGTGTAC964    GluSerAlaHisAspThrAspGlyAsnLeuSerIleAspArgValTyr    300305310    AACACACATAGGCAGAGTGTTAACCATGCTGACAGCATCTGGAACTTT1012    AsnThrHisArgGlnSerValAsnHisAlaAspSerIleTrpAsnPhe    315320325    CATTGTTGGATCGAGTCTTACATGCAGAGAGAAGATCTACCTGAAGGA1060    HisCysTrpIleGluSerTyrMetGlnArgGluAspLeuProGluGly    330335340    TATGGTGGCTGGCAAGTCTTGGACCCCACACCTCAGGAGAGGAGTAGT1108    TyrGlyGlyTrpGlnValLeuAspProThrProGlnGluArgSerSer    345350355    GGTATGTTTCGCTGTGGCCCATGTCCATTGAAGGCCATTAAAGAAGGG1156    GlyMetPheArgCysGlyProCysProLeuLysAlaIleLysGluGly    360365370375    GACCTCAATGTGAAGTTTGATGTTCCATTTGTCTTTGCTGAGGTGAAT1204    AspLeuAsnValLysPheAspValProPheValPheAlaGluValAsn    380385390    GCAGACATCATCAATTGGGAAATCAGACCAGACGGTCAGCGAATGCGG1252    AlaAspIleIleAsnTrpGluIleArgProAspGlyGlnArgMetArg    395400405    CTTTCATCCAACTCCGCAAAAGTGGGGAGGAACATTAGCACCAAAAGT1300    LeuSerSerAsnSerAlaLysValGlyArgAsnIleSerThrLysSer    410415420    CCTTACAGTAACGAGAGGGAAGATATAACCCTTCAGTACAAGTACCAA1348    ProTyrSerAsnGluArgGluAspIleThrLeuGlnTyrLysTyrGln    425430435    GAAGGTTCAGCCAAGGAGCGGGAGGTGTACAACAAGGCAGGGCGGCGC1396    GluGlySerAlaLysGluArgGluValTyrAsnLysAlaGlyArgArg    440445450455    ATCTCCGGGCCGGATAGAGAAGAGGAATCAAAACCAGCCAATGAACCA1444    IleSerGlyProAspArgGluGluGluSerLysProAlaAsnGluPro    460465470    GGAAACGTGCAGCTGGAGATCAGATACGCCAAGCCTGTGTTCGGGACC1492    GlyAsnValGlnLeuGluIleArgTyrAlaLysProValPheGlyThr    475480485    GACTTTGACGTCATCTTTGAGTTGGAGAACATGGGAGACGAAGAAGTC1540    AspPheAspValIlePheGluLeuGluAsnMetGlyAspGluGluVal    490495500    AGCTGCAAATTGAACATGATGTCAAAGGCTGTCACGTATAACTCGGTC1588    SerCysLysLeuAsnMetMetSerLysAlaValThrTyrAsnSerVal    505510515    CACCTGGGAGAGTGCCAGAATAGCACAGTCAATGTTGTCATTCCTGCT1636    HisLeuGlyGluCysGlnAsnSerThrValAsnValValIleProAla    520525530535    CACAAAGTCCACAGGGAGACGGTGCGTCTACTCTACACTAAGTATGCA1684    HisLysValHisArgGluThrValArgLeuLeuTyrThrLysTyrAla    540545550    TCGTGCGTCAGCGAACACAACATCATCCGGGTGGTAGGGGTGGCAAGA1732    SerCysValSerGluHisAsnIleIleArgValValGlyValAlaArg    555560565    GTGTCCGGCCAGGAAAAATCCATCCTGGAGATGGTCAACATCCCACTG1780    ValSerGlyGlnGluLysSerIleLeuGluMetValAsnIleProLeu    570575580    AGCAAGCCCAAACTCAGTATTAAGGTTCCTGGCTGGGTGATTTTAAAT1828    SerLysProLysLeuSerIleLysValProGlyTrpValIleLeuAsn    585590595    AGGAAAATCACCACCGTCATCACCTTCACCAATCCATTGCCAGTGCCA1876    ArgLysIleThrThrValIleThrPheThrAsnProLeuProValPro    600605610615    CTGAACCGAGGAGTGTTCACTGTTGAAGGGGCTGGCCTACTTTCAACC1924    LeuAsnArgGlyValPheThrValGluGlyAlaGlyLeuLeuSerThr    620625630    AAAGAGATCCGCATTTCTGGTAGCATCGCTCCAGGCCAGCGTGTGTCT1972    LysGluIleArgIleSerGlySerIleAlaProGlyGlnArgValSer    635640645    GTGGAGCTGTCCTTCACACCCATGAGGGCGGGGGTCAGGGAGTTCCTG2020    ValGluLeuSerPheThrProMetArgAlaGlyValArgGluPheLeu    650655660    GTGGACTTTGACTCCGACAGGCTCCAGGACGTGAAGGGAGTCGCCACA2068    ValAspPheAspSerAspArgLeuGlnAspValLysGlyValAlaThr    665670675    CTGGTGGTCCGCAAGACTTCACCCTCCTATTTTCCCATGCCCTACACG2116    LeuValValArgLysThrSerProSerTyrPheProMetProTyrThr    680685690695    TTGTGATCAAACCTATAGCTGTCAACAGGGCTCTGGCACTCATTCTTATACTA2169    Leu    ACAAATATATTTAGCAAAGTCAAGCAAGGGTTTCACTTTTCTTAATATACCATGATGTGT2229    AGCGCTGATTCAATTAATGAATAAATTAATTTCAATTAATGTGAAGAAAATGCAAACATT2289    GCCTTAATTCTTTGCAATGTCACAGGAATAGCGTAAATCATGGCTCATTGATATTAAATG2349    TAGTATTGACATATATCCATGCATTTTGCACTTCTGCAAATCACCATTTTGTTGTTAATC2409    AATGTTTTACCACGATTTTTGCATCTATTCTTGTTTAATTGTAATCAAGACATTTACATG2469    ATTGTGGGGGCCAAAGTATATAGATGTTGTGGTTGGGAAATGGGGCAATAATAGGGGAAG2529    GGTTAATTATAGGGTCAGTGTTAGTAATTGGTTAAGGTTACTAATAGGGTAAGTGTTACA2589    GTGTAAAGATAAGCCTTTGATTTTGTTAAATTTATTATGCCTTTCATCAACAGTGGTTTG2649    GGGTTTTATAACAACAATTAAAGTGCTTAACTACTGGTGAACGACGTTGCAGAACGTATA2709    TGGTACAAGTTTGTGTTGATCGCATGGAAAAGGGAATAACCAGTTACAACTTATATGGTA2769    AGAGCCTGGTAATACCATGGAAACAAACGAGGCTTCCTTTTACAGTACAGTTTCAGCGTC2829    ATGAATATTTGGCCTGTTAAGCCCTTTGAGACTGTAATGGTGATTAAGGGCTATACAAAT2889    AAAATTGAATTGAATTGAATTAAAAAAAAAAA2921    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 696 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    MetAlaHisThrAsnArgLeuIleAlaGlyValAspLeuArgSerGln    151015    GluAsnAsnArgGluHisArgThrGluGluIleAspArgLysArgLeu    202530    IleValArgArgGlyGlnAlaPheSerLeuThrValHisLeuSerAsp    354045    ProLeuGlnSerGlyHisGluLeuAlaLeuValLeuLysGlnAspLys    505560    AsnAsnAspAspIleValIleArgGlnArgThrAlaGlyGlySerGly    65707580    AspLysTrpTrpLeuHisGlnGlnSerAlaArgAsnGluLeuLeuLeu    859095    ThrValTyrSerProAlaArgAlaAlaValGlyGluTyrArgLeuAla    100105110    ValGluLeuMetSerGlyAsnLysLeuLeuGluArgThrAspPheThr    115120125    LysMetTyrLeuLeuPheAsnProTrpCysLysAspAspAlaValTyr    130135140    LeuProAspGluSerLeuLeuLysGluTyrIleMetAsnGluAsnGly    145150155160    ArgIlePheThrGlySerAlaAspTrpMetSerGlyLeuProTrpAsn    165170175    PheGlyGlnPheGluAspAsnValMetAspIleCysPheGluIleLeu    180185190    AspArgPheLysProAlaArgSerAspProProAsnAspMetArgGln    195200205    ArgTrpAspProValTyrIleSerArgAlaValValAlaMetValAsn    210215220    AlaAsnAspAspGlyGlyValLeuValGlyLysTrpGlnGluProTyr    225230235240    ThrGlyGlyValGlnProThrLysTrpMetSerSerValProIleLeu    245250255    GluLysTrpSerLysSerLysSerGlyValLysTyrGlyGlnCysTrp    260265270    ValPheAlaAlaValAlaCysThrValLeuArgCysLeuGlyIlePro    275280285    ThrArgCysIleThrAsnPheGluSerAlaHisAspThrAspGlyAsn    290295300    LeuSerIleAspArgValTyrAsnThrHisArgGlnSerValAsnHis    305310315320    AlaAspSerIleTrpAsnPheHisCysTrpIleGluSerTyrMetGln    325330335    ArgGluAspLeuProGluGlyTyrGlyGlyTrpGlnValLeuAspPro    340345350    ThrProGlnGluArgSerSerGlyMetPheArgCysGlyProCysPro    355360365    LeuLysAlaIleLysGluGlyAspLeuAsnValLysPheAspValPro    370375380    PheValPheAlaGluValAsnAlaAspIleIleAsnTrpGluIleArg    385390395400    ProAspGlyGlnArgMetArgLeuSerSerAsnSerAlaLysValGly    405410415    ArgAsnIleSerThrLysSerProTyrSerAsnGluArgGluAspIle    420425430    ThrLeuGlnTyrLysTyrGlnGluGlySerAlaLysGluArgGluVal    435440445    TyrAsnLysAlaGlyArgArgIleSerGlyProAspArgGluGluGlu    450455460    SerLysProAlaAsnGluProGlyAsnValGlnLeuGluIleArgTyr    465470475480    AlaLysProValPheGlyThrAspPheAspValIlePheGluLeuGlu    485490495    AsnMetGlyAspGluGluValSerCysLysLeuAsnMetMetSerLys    500505510    AlaValThrTyrAsnSerValHisLeuGlyGluCysGlnAsnSerThr    515520525    ValAsnValValIleProAlaHisLysValHisArgGluThrValArg    530535540    LeuLeuTyrThrLysTyrAlaSerCysValSerGluHisAsnIleIle    545550555560    ArgValValGlyValAlaArgValSerGlyGlnGluLysSerIleLeu    565570575    GluMetValAsnIleProLeuSerLysProLysLeuSerIleLysVal    580585590    ProGlyTrpValIleLeuAsnArgLysIleThrThrValIleThrPhe    595600605    ThrAsnProLeuProValProLeuAsnArgGlyValPheThrValGlu    610615620    GlyAlaGlyLeuLeuSerThrLysGluIleArgIleSerGlySerIle    625630635640    AlaProGlyGlnArgValSerValGluLeuSerPheThrProMetArg    645650655    AlaGlyValArgGluPheLeuValAspPheAspSerAspArgLeuGln    660665670    AspValLysGlyValAlaThrLeuValValArgLysThrSerProSer    675680685    TyrPheProMetProTyrThrLeu    690695    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 29 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    GTCAAGTACGGCCAGTGCTGGGTCTTCGC29    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    AATTCATCGATTAGTAAGGAGGTTTAAAAT30    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    GGCTTCTTATAAAGGTCTGATTGTTGATGT30    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 32 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    TAATGGTCGTTCTCATGAAAACAACCTGGCAC32    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    ATCGTACGCGTGAAATCGACCGTGAGCGCCTGA33    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    AGCCATTTTAAACCTCCTTACTAATCGATG30    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    ATTAACATCAACAATCAGACCTTTATAAGA30    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 32 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    CGATGTGCCAGGTTGTTTTCATGAGAACGACC32    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 33 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    AGCTTCAGGCGCTCACGGTCGATTTCACGCGTA33    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Pagrus major    (F) TISSUE TYPE: liver    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    ValLysTyrGlyGlnCysTrpValPheAlaAlaValAlaCysThrVal    151015    LeuArgCysLeuGlyIleProThrArg    2025    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 30 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    TTGGAAGCTTGTAAGAGCAACTCTTGGAAA30    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    TTGTACACTCGATCGATGGAGAGGT25    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 24 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    TCTGCTTTGGGATCCTTGACCGCT24    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 23 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    TGAAGGAGAGCTCCACAGACACA23    (2) INFORMATION FOR SEQ ID NO:25:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    ATGATGTCAAAGGCTGTCAC20    (2) INFORMATION FOR SEQ ID NO:26:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    TCTTACCATATAAGTTGTAA20    (2) INFORMATION FOR SEQ ID NO:27:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 20 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    ATTGATTAACAACAAAATGG20    (2) INFORMATION FOR SEQ ID NO:28:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1921 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: unknown    (D) TOPOLOGY: unknown    (ii) MOLECULE TYPE: DNA (genomic)    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Theragra chalcogramma    (F) TISSUE TYPE: muscle    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    GCCCACACAAACCGTTTAATTGCTGGTGTTGATCTGAGAAGCCAGGAAAACAACCGGGAA60    CACCGAACTGAGGAGATTGATAGGAAGCGTTTGATTGTTCGGCGGGGACAAGCCTTCTCC120    CTGACGGTGCACCTCTCCGACCCGCTGCAGTCCGGCCATGAGCTGGCCCTGGTCTTAAAG180    CAGGATAAAATCAACGATGATATTGTGATCAGACAGCGAACGACTGGAGGGTCCGGTGAC240    AAGTGGTGGTTACACCAGCAGAGCGCGAACAACGAATTACTGCTGACTGTGTACAGTCCC300    GCCCGTGCTGCCGTTGGCGAGTACCGCTTGGCTGTTGAACTGATGTCAGGGAATAAACTT360    CTGGAGAGGACGGACTTTACCAAAATGTACTTGCTGTTTAATCCCTGGTGCAAAGAAGAT420    GCCGTGTACCTCCCTGATGAGTGTCTGCTCAAGGAATACATTATGAACGAGAATGGTCGC480    ATTTTCACTGGGAGTGCGGATTGGATGAGTGGGTTGCCATGGAATTTCGGACAGTTTGAA540    GATAATGTGATGGACATCTGCTTTGAGATCCTTGACCGCTTTAACCCAGCGAGGTCAGAC600    CCCCCAAGCGACATGCTTCAGCGATGGGACCCTGTCTACATCAGCAGGGCAGTCGTTGCC660    ATGGTGAATGCCAACGATGATGACGGTGGAGTCGTGGTGGGTCGATGGCAGGAACCTTAC720    ACAGGTGGAGTACAGCCAACCAAATGGATGAGCAGTGTGCCCATCCTGGAAGAGTGGAGC780    AAATCAAAGTCTGGAGTGAAATATGGCCAATGCTGGGTGTTTGCAGCCGTGGCCTGCACA840    GTGATGCGATGCCTGGGCATCCCCACACGCTGCATCACCAACTTTCAGTCGGCCCATGAC900    ACAGACGGAAACCTCTCCATCGACCGAGTGTACAACATACATAGGCAGCTAGTTGACGGT960    GATGACAGTATCTGGAACTTTCATTGTTGGATCGAGTCTTACATGCAGAGAGAAGATCTA1020    CCTGAAGGATATGGTGGCTGGCAAGTCTTGGACCCCACACCTCAGGAGAGGAGTAGTGGT1080    ATGTTTCGCTGTGGCCCATGTCCTTTGAAGGCCATTAAAGAAGGGGACCTCAATGTGAAG1140    TTTGATGTTCCATTTGTCTTTGCTGAGGTGAATGCAGACATCATCAATTGGGAAATCAGA1200    CCAGACGGTCAGCGAAAGCGGCTTTCATCCAACTCTGCAAATGTGGGGAGGAACATTAGC1260    ACCAAAAGTCCTTATGGTAACGAGAGGGAAGATATAACCCATCAGTACAAGTACCAAGAA1320    GGTTCAGCCAAGGAGCGGGAGGTGTACAACAAGGCAGGGCGGCGCATCTCCGGGCCGGAT1380    GGAGAAGAGGAATCAAAACCAGGAAACGTGCAGCTGGAGATCAAGCACGCCAAACCTGTG1440    TTCGGGACCGACTTTGACGTCATCTTTGAGTTGGAGAACATGGGAGACAAAGAAGTCAGC1500    TGCAAATTAAACATGATGTCAGAGGCTGTCACCTATAACTCAGTTCACCTTGGACGGTTC1560    CAGAACAGCACGGTCAATGTTGTCATTCCTGCTCACAAAGTCCACAGTGAGACGGTGCGT1620    CTACTCTACACTAAGTATGCCTCAGTTGTCAGCGAGCACAACATCATCCGGGTGACAGGG1680    GTGGCGGAAGTGTCCGGCCAGGAAAAATCCATCCTGGAGATGGTCAACATCCCACTGAGC1740    AAGCCCAAACTCAGTATTAAGGTTCCTGGCTGGGTGATTTTAAATAGGAAAATCACCACC1800    TTCATCTCCTTCACCAATCCATTGCCAGTGCCACTGAACCGAGGAGTGTTCACTGTTGAA1860    GGGGCTGGCCTACTTCCCACCAAAGAGATCCGCATTTCTGGTAGCATCGCTCCAGGCCAG1920    C1921    (2) INFORMATION FOR SEQ ID NO:29:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (genomic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    ACACTGCCGGTCCATCGAA19    (2) INFORMATION FOR SEQ ID NO:30:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2064 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA to mRNA    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Paralichthys olivaceus    (F) TISSUE TYPE: liver    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..2061    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    GACAATCAGAACATTCCGATCACTGATGTGGATGTGAGAAGTCATGAA48    AspAsnGlnAsnIleProIleThrAspValAspValArgSerHisGlu    151015    AACAACTTGGCTCACCGCACCAGGGAGATTGATCGGGAGCGCTTGATC96    AsnAsnLeuAlaHisArgThrArgGluIleAspArgGluArgLeuIle    202530    GTCCGCAGGGGTCAACCCTTCTCCATATCTCTGCAGTGCTGCGACTCG144    ValArgArgGlyGlnProPheSerIleSerLeuGlnCysCysAspSer    354045    CTGACCCGGAATCACCATCTGGAACTGTCCCTGCACCTCGGTAAGAAA192    LeuThrArgAsnHisHisLeuGluLeuSerLeuHisLeuGlyLysLys    505560    GATGAGGTGGTGATTAAGGTGCACAATGAGCCTGAGGCTGGAGGCAAG240    AspGluValValIleLysValHisAsnGluProGluAlaGlyGlyLys    65707580    TGGTGGTTTAACCATCAGAAAGTGCAGGATGAAATTCTGCTGACTCTA288    TrpTrpPheAsnHisGlnLysValGlnAspGluIleLeuLeuThrLeu    859095    CACAGTCCAGCGGACGCCATAATTGGCGAGTACCACCTGACTGTGTTG336    HisSerProAlaAspAlaIleIleGlyGluTyrHisLeuThrValLeu    100105110    ATCAAGTCACCGGATGGACACTTTGTGAAGAAGACTAAGAACATTGGA384    IleLysSerProAspGlyHisPheValLysLysThrLysAsnIleGly    115120125    TTCCACCTGCTCTTTAACCCCTGGTGCAAAGATGATGCTGTGTACCTC432    PheHisLeuLeuPheAsnProTrpCysLysAspAspAlaValTyrLeu    130135140    CCTGATGAAAGGATGCTCGACGAGTATGTTATGAATGAGGAGGGGATC480    ProAspGluArgMetLeuAspGluTyrValMetAsnGluGluGlyIle    145150155160    ATTTACAGGGGAACCTCGAATCACATCAGTAGCATACCCTGGAATTAC528    IleTyrArgGlyThrSerAsnHisIleSerSerIleProTrpAsnTyr    165170175    GGACAGTTTGAGGACTATGTGATGGACATCTGTTTTCAAGTTCTGGAC576    GlyGlnPheGluAspTyrValMetAspIleCysPheGlnValLeuAsp    180185190    AACTCCAAGGAAGCCCTGAAGAATTCAAAGATGGACATTGAGAAGAGA624    AsnSerLysGluAlaLeuLysAsnSerLysMetAspIleGluLysArg    195200205    TCTGACCCTGTCTATGTCAGCAGGATGATCACTGCGATGGTGAACTCT672    SerAspProValTyrValSerArgMetIleThrAlaMetValAsnSer    210215220    AACGGTGACAGGGGTGTGCTGACTGGTCAGTGGCACGAGCCATACACT720    AsnGlyAspArgGlyValLeuThrGlyGlnTrpHisGluProTyrThr    225230235240    GGCGGGTTCTCACCACTTCGATGGACCGGCAGCGTGCCCATCCTCCGG768    GlyGlyPheSerProLeuArgTrpThrGlySerValProIleLeuArg    245250255    AAGTGGAGCAAGGCCGAGGTCAGGGCGGTCAAATATGGCCAGTGCTGG816    LysTrpSerLysAlaGluValArgAlaValLysTyrGlyGlnCysTrp    260265270    GTGTTTGCTGCTGTCGCCTGCACAGTGCTGCGTTGTCTGGGAATCCCA864    ValPheAlaAlaValAlaCysThrValLeuArgCysLeuGlyIlePro    275280285    ACACGCAACATCACTAACTTCAATTCAGCACATGATGTCGATGGAAAC912    ThrArgAsnIleThrAsnPheAsnSerAlaHisAspValAspGlyAsn    290295300    CTCTCCGTCGACATCGTGTTGAACAAAGAAATGGAGAGCGTTGGCAAG960    LeuSerValAspIleValLeuAsnLysGluMetGluSerValGlyLys    305310315320    AAGGACAGTAGCTGGAACTTCCACTGTTGGATCGAGTCCTGGATGAGG1008    LysAspSerSerTrpAsnPheHisCysTrpIleGluSerTrpMetArg    325330335    AGAGACGACCTCTCTAAAGGAAATGACGGCTGGCAGGTTTTGGACCCC1056    ArgAspAspLeuSerLysGlyAsnAspGlyTrpGlnValLeuAspPro    340345350    ACCCCTCAAGAACTGAGTGATGGTGAGTATTGCTGCGGCCCGTGTCCA1104    ThrProGlnGluLeuSerAspGlyGluTyrCysCysGlyProCysPro    355360365    GTCACCGCCATCAAGGAGGGAAATCTGAGTGTGAAGTACGACGCTCCG1152    ValThrAlaIleLysGluGlyAsnLeuSerValLysTyrAspAlaPro    370375380    TTTATCTTCGCTGAGGTGAACGCTGACATCATCTACTGGATGGCTGGA1200    PheIlePheAlaGluValAsnAlaAspIleIleTyrTrpMetAlaGly    385390395400    CCAGGAGGCGAACGGAAGAAGATCGATGTGGACCAGAGTGGTGTGGGG1248    ProGlyGlyGluArgLysLysIleAspValAspGlnSerGlyValGly    405410415    AAGAACATCAGCACCAAAAGTCTTTATGGCGACTACAGGGAGGATGTC1296    LysAsnIleSerThrLysSerLeuTyrGlyAspTyrArgGluAspVal    420425430    ACTCTGCACTACAAATACCCCGAAGGCTCCAAGAAGGAGAGAGAGGTG1344    ThrLeuHisTyrLysTyrProGluGlySerLysLysGluArgGluVal    435440445    TACCAGAAGGCCGGACACCGAATCAAAGAGCAGATCTGTGAAAACAAA1392    TyrGlnLysAlaGlyHisArgIleLysGluGlnIleCysGluAsnLys    450455460    GGTCCACAACAACTGCAGCTGTCAGTCAAGCACGGGAAACCTGTATTT1440    GlyProGlnGlnLeuGlnLeuSerValLysHisGlyLysProValPhe    465470475480    GGCACTGACTTCGATGTGATAGTTGAGGTGAAGAATGAAGGACAGAAA1488    GlyThrAspPheAspValIleValGluValLysAsnGluGlyGlnLys    485490495    GACACCAGTCCACAGCTGCTGATTGTGGTCATGGCCGTGACCTACAAT1536    AspThrSerProGlnLeuLeuIleValValMetAlaValThrTyrAsn    500505510    TCCATCAATCAAGGGGAGTGTCAGAGGAAGGCGACCATAGTGACCGTG1584    SerIleAsnGlnGlyGluCysGlnArgLysAlaThrIleValThrVal    515520525    CCGGCTCGCAAAACCCACAAGGAAGTGCTGCGTCTGCGCTACGACGAC1632    ProAlaArgLysThrHisLysGluValLeuArgLeuArgTyrAspAsp    530535540    TATGTCAAATGTGTCTCTGAGCACCATCTGATCAGGGTGAAAGCGCTC1680    TyrValLysCysValSerGluHisHisLeuIleArgValLysAlaLeu    545550555560    ATGGAGGTTCCAGGGGACAACAAACCCGTCATGAGTGTGGCCAACATT1728    MetGluValProGlyAspAsnLysProValMetSerValAlaAsnIle    565570575    CCACTGAGCATGCCTGAGCTCCTGGTAGAGGTACCTGGGAGCATCATT1776    ProLeuSerMetProGluLeuLeuValGluValProGlySerIleIle    580585590    GTTCAGGAGAAGGTGACAGCCTTCATCTCCTTCACAAATCCTCTAACT1824    ValGlnGluLysValThrAlaPheIleSerPheThrAsnProLeuThr    595600605    GTCCCACTGAAGCGTGGCATGTTCACCGTTGAGGGGTCCGGACTACTG1872    ValProLeuLysArgGlyMetPheThrValGluGlySerGlyLeuLeu    610615620    TCTGCCTCTGAGATCTATGTGAAAGGGGACATTGCTCCAGGCCAGAAG1920    SerAlaSerGluIleTyrValLysGlyAspIleAlaProGlyGlnLys    625630635640    GTTTCTGTCAAGATCACCTTCACGCCCATGAGGGTCGGGGTGAGGAAG1968    ValSerValLysIleThrPheThrProMetArgValGlyValArgLys    645650655    CTCCTGGTGGACTTTGACTCTGACAGGCTGAAGGATGTGAAAGGAGTC2016    LeuLeuValAspPheAspSerAspArgLeuLysAspValLysGlyVal    660665670    ACGACAGTGGTCGTCCGCAAGAAATCCTGTTTTATTAGGTGTCCT2061    ThrThrValValValArgLysLysSerCysPheIleArgCysPro    675680685    TAA2064    (2) INFORMATION FOR SEQ ID NO:31:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 687 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    AspAsnGlnAsnIleProIleThrAspValAspValArgSerHisGlu    151015    AsnAsnLeuAlaHisArgThrArgGluIleAspArgGluArgLeuIle    202530    ValArgArgGlyGlnProPheSerIleSerLeuGlnCysCysAspSer    354045    LeuThrArgAsnHisHisLeuGluLeuSerLeuHisLeuGlyLysLys    505560    AspGluValValIleLysValHisAsnGluProGluAlaGlyGlyLys    65707580    TrpTrpPheAsnHisGlnLysValGlnAspGluIleLeuLeuThrLeu    859095    HisSerProAlaAspAlaIleIleGlyGluTyrHisLeuThrValLeu    100105110    IleLysSerProAspGlyHisPheValLysLysThrLysAsnIleGly    115120125    PheHisLeuLeuPheAsnProTrpCysLysAspAspAlaValTyrLeu    130135140    ProAspGluArgMetLeuAspGluTyrValMetAsnGluGluGlyIle    145150155160    IleTyrArgGlyThrSerAsnHisIleSerSerIleProTrpAsnTyr    165170175    GlyGlnPheGluAspTyrValMetAspIleCysPheGlnValLeuAsp    180185190    AsnSerLysGluAlaLeuLysAsnSerLysMetAspIleGluLysArg    195200205    SerAspProValTyrValSerArgMetIleThrAlaMetValAsnSer    210215220    AsnGlyAspArgGlyValLeuThrGlyGlnTrpHisGluProTyrThr    225230235240    GlyGlyPheSerProLeuArgTrpThrGlySerValProIleLeuArg    245250255    LysTrpSerLysAlaGluValArgAlaValLysTyrGlyGlnCysTrp    260265270    ValPheAlaAlaValAlaCysThrValLeuArgCysLeuGlyIlePro    275280285    ThrArgAsnIleThrAsnPheAsnSerAlaHisAspValAspGlyAsn    290295300    LeuSerValAspIleValLeuAsnLysGluMetGluSerValGlyLys    305310315320    LysAspSerSerTrpAsnPheHisCysTrpIleGluSerTrpMetArg    325330335    ArgAspAspLeuSerLysGlyAsnAspGlyTrpGlnValLeuAspPro    340345350    ThrProGlnGluLeuSerAspGlyGluTyrCysCysGlyProCysPro    355360365    ValThrAlaIleLysGluGlyAsnLeuSerValLysTyrAspAlaPro    370375380    PheIlePheAlaGluValAsnAlaAspIleIleTyrTrpMetAlaGly    385390395400    ProGlyGlyGluArgLysLysIleAspValAspGlnSerGlyValGly    405410415    LysAsnIleSerThrLysSerLeuTyrGlyAspTyrArgGluAspVal    420425430    ThrLeuHisTyrLysTyrProGluGlySerLysLysGluArgGluVal    435440445    TyrGlnLysAlaGlyHisArgIleLysGluGlnIleCysGluAsnLys    450455460    GlyProGlnGlnLeuGlnLeuSerValLysHisGlyLysProValPhe    465470475480    GlyThrAspPheAspValIleValGluValLysAsnGluGlyGlnLys    485490495    AspThrSerProGlnLeuLeuIleValValMetAlaValThrTyrAsn    500505510    SerIleAsnGlnGlyGluCysGlnArgLysAlaThrIleValThrVal    515520525    ProAlaArgLysThrHisLysGluValLeuArgLeuArgTyrAspAsp    530535540    TyrValLysCysValSerGluHisHisLeuIleArgValLysAlaLeu    545550555560    MetGluValProGlyAspAsnLysProValMetSerValAlaAsnIle    565570575    ProLeuSerMetProGluLeuLeuValGluValProGlySerIleIle    580585590    ValGlnGluLysValThrAlaPheIleSerPheThrAsnProLeuThr    595600605    ValProLeuLysArgGlyMetPheThrValGluGlySerGlyLeuLeu    610615620    SerAlaSerGluIleTyrValLysGlyAspIleAlaProGlyGlnLys    625630635640    ValSerValLysIleThrPheThrProMetArgValGlyValArgLys    645650655    LeuLeuValAspPheAspSerAspArgLeuLysAspValLysGlyVal    660665670    ThrThrValValValArgLysLysSerCysPheIleArgCysPro    675680685    (2) INFORMATION FOR SEQ ID NO:32:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2064 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA to mRNA    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Paralichthys olivaceus    (F) TISSUE TYPE: liver    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 1..2061    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    GACAATCAGAACATTCCGATCACTGATGTGGATGTGAGAAGTCATGAA48    AspAsnGlnAsnIleProIleThrAspValAspValArgSerHisGlu    151015    AACAACTTGGCTCACCGCACCAGGGAGATTGATCGGGAGCGCTTGATC96    AsnAsnLeuAlaHisArgThrArgGluIleAspArgGluArgLeuIle    202530    GTCCGCAGGGGTCAACCCTTCTCCATATCTCTGCAGTGCTGCGACTCG144    ValArgArgGlyGlnProPheSerIleSerLeuGlnCysCysAspSer    354045    CTGACCCGGAATCACCATCTGGAACTGTCCCTGCACCTCGGTAAGAAA192    LeuThrArgAsnHisHisLeuGluLeuSerLeuHisLeuGlyLysLys    505560    GATGAGGTGGTGATTAAGGTGCACAATGAGCCTGAGGCTGGAGGCAAG240    AspGluValValIleLysValHisAsnGluProGluAlaGlyGlyLys    65707580    TGGTGGTTTAACCATCAGAAAGTGCAGGATGAAATTCTGCTGACTCTA288    TrpTrpPheAsnHisGlnLysValGlnAspGluIleLeuLeuThrLeu    859095    CACAGTCCAGCGGACGCCATAATTGGCGAGTACCACCTGACTGTGTTG336    HisSerProAlaAspAlaIleIleGlyGluTyrHisLeuThrValLeu    100105110    ATCAAGTCACCGGATGGACACTTTGTGAAGAAGACTAAGAACATTGGA384    IleLysSerProAspGlyHisPheValLysLysThrLysAsnIleGly    115120125    TTCCACCTGCTCTTTAACCCCTGGTGCAAAGATGATGCTGTGTACCTC432    PheHisLeuLeuPheAsnProTrpCysLysAspAspAlaValTyrLeu    130135140    CCTGATGAAAGGATGCTCGACGAGTATGTTATGAATGAGGAGGGGATC480    ProAspGluArgMetLeuAspGluTyrValMetAsnGluGluGlyIle    145150155160    ATTTACAGGGGAACCTCGAATCACATCAGTAGCATACCCTGGAATTAC528    IleTyrArgGlyThrSerAsnHisIleSerSerIleProTrpAsnTyr    165170175    GGACAGTTTGAGGACTATGTGATGGACATCTGTTTTCAAGTTCTGGAC576    GlyGlnPheGluAspTyrValMetAspIleCysPheGlnValLeuAsp    180185190    AACTCCAAGGAAGCCCTGAAGAATTCAAAGATGGACATTGAGAAGAGA624    AsnSerLysGluAlaLeuLysAsnSerLysMetAspIleGluLysArg    195200205    TCTGACCCTGTCTATGTCAGCAGGATGATCACTGCGATGGTGAACTCT672    SerAspProValTyrValSerArgMetIleThrAlaMetValAsnSer    210215220    AACGGTGACAGGGGTGTGCTGACTGGTCAGTGGCACGAGCCATACACT720    AsnGlyAspArgGlyValLeuThrGlyGlnTrpHisGluProTyrThr    225230235240    GGCGGGTTCTCACCACTTCGATGGACCGGCAGCGTGCCCATCCTCCGG768    GlyGlyPheSerProLeuArgTrpThrGlySerValProIleLeuArg    245250255    AAGTGGAGCAAGGCCGAGGTCAGGGCGGTCAAATATGGCCAGTGCTGG816    LysTrpSerLysAlaGluValArgAlaValLysTyrGlyGlnCysTrp    260265270    GTGTTTGCTGCTGTCGCCTGCACAGTGCTGCGTTGTCTGGGAATCCCA864    ValPheAlaAlaValAlaCysThrValLeuArgCysLeuGlyIlePro    275280285    ACACGCAACATCACTAACTTCAATTCAGCACATGATGTCGATGGAAAC912    ThrArgAsnIleThrAsnPheAsnSerAlaHisAspValAspGlyAsn    290295300    CTCTCCGTCGACATCGTGTTGAACAAAGAAATGGAGAGCGTTGGCAAG960    LeuSerValAspIleValLeuAsnLysGluMetGluSerValGlyLys    305310315320    AAGGACAGTAGCTGGAACTTCCACTGTTGGATCGAGTCCTGGATGAGG1008    LysAspSerSerTrpAsnPheHisCysTrpIleGluSerTrpMetArg    325330335    AGAGACGACCTCTCTAAAGGAAATGACGGCTGGCAGGTTTTGGACCCC1056    ArgAspAspLeuSerLysGlyAsnAspGlyTrpGlnValLeuAspPro    340345350    ACCCCTCAAGAACTGAGTGATGGTGAGTATTGCTGCGGCCCGTGTCCA1104    ThrProGlnGluLeuSerAspGlyGluTyrCysCysGlyProCysPro    355360365    GTCACCGCCATCAAGGAGGGAAATCTGAGTGTGAAGTACGACGCTCCG1152    ValThrAlaIleLysGluGlyAsnLeuSerValLysTyrAspAlaPro    370375380    TTTATCTTCGCTGAGGTGAACGCTGACATCATCTACTGGATGGCTGGA1200    PheIlePheAlaGluValAsnAlaAspIleIleTyrTrpMetAlaGly    385390395400    CCAGGAGGCGAACGGAAGAAGATCGATGTGGACCAGAGTGGTGTGGGG1248    ProGlyGlyGluArgLysLysIleAspValAspGlnSerGlyValGly    405410415    AAGAACATCAGCACCAAAAGTCTTTATGGCGACTACAGGGAGGATGTC1296    LysAsnIleSerThrLysSerLeuTyrGlyAspTyrArgGluAspVal    420425430    ACTCTGCACTACAAATACCCCGAAGGCTCCAAGAAGGAGAGAGAGGTG1344    ThrLeuHisTyrLysTyrProGluGlySerLysLysGluArgGluVal    435440445    TACCAGAAGGCCGGACACCGAATCAAAGAGCAGATCTGTGAAAACAAA1392    TyrGlnLysAlaGlyHisArgIleLysGluGlnIleCysGluAsnLys    450455460    GGTCCACAACAACTGCAGCTGTCAGTCAAGCACGGGAAACCTGTATTT1440    GlyProGlnGlnLeuGlnLeuSerValLysHisGlyLysProValPhe    465470475480    GGCACTGACTTCGATGTGATAGTTGAGGTGAAGAATGAAGGACAGAAA1488    GlyThrAspPheAspValIleValGluValLysAsnGluGlyGlnLys    485490495    GACACCAGTCCACAGCTGCTGATTGTGGTCATGGCCGTGACCTACAAT1536    AspThrSerProGlnLeuLeuIleValValMetAlaValThrTyrAsn    500505510    TCCATCAATCAAGGGGAGTGTCAGAGGAAGGCGACCATAGTGACCGTG1584    SerIleAsnGlnGlyGluCysGlnArgLysAlaThrIleValThrVal    515520525    CCGGCTCGCAAAACCCACAAGGAAGTGCTGCGTCTGCGCTACGACGAC1632    ProAlaArgLysThrHisLysGluValLeuArgLeuArgTyrAspAsp    530535540    TATGTCAAATGTGTCTCTGAGCACCATCTGATCAGGGTGAAAGCGCTC1680    TyrValLysCysValSerGluHisHisLeuIleArgValLysAlaLeu    545550555560    ATGGAGGTTCCAGGGGACAACAAACCCGTCATGAGTGTGGCCAACATT1728    MetGluValProGlyAspAsnLysProValMetSerValAlaAsnIle    565570575    CCACTGAGCATGCCTGAGCTCCTGGTAGAGGTACCTGGGAGCATCATT1776    ProLeuSerMetProGluLeuLeuValGluValProGlySerIleIle    580585590    GTTCAGGAGAAGGTGACAGCCTTCATCTCCTTCACAAATCCTCTAACT1824    ValGlnGluLysValThrAlaPheIleSerPheThrAsnProLeuThr    595600605    GTCCCACTGAAGCGTGGCATGTTCACCGTGGAGGGGTCCGGACTACTG1872    ValProLeuLysArgGlyMetPheThrValGluGlySerGlyLeuLeu    610615620    TCTGCCTCTGAGATCTATGTGAAAGGGGACATTGCTCCAGGCCAGAAG1920    SerAlaSerGluIleTyrValLysGlyAspIleAlaProGlyGlnLys    625630635640    GTTTCTGTCAAGATCACCTTCACGCCCATGAGGGTCGGGGTGAGGAAG1968    ValSerValLysIleThrPheThrProMetArgValGlyValArgLys    645650655    CTCCTGGTGGACTTTGACTCTGACAGGCTGAAGGATGTGAAAGGAGTC2016    LeuLeuValAspPheAspSerAspArgLeuLysAspValLysGlyVal    660665670    ACGACAGTGGTCGTCCGCAAGAAATCCTGTTTTATTAGGTGTCCT2061    ThrThrValValValArgLysLysSerCysPheIleArgCysPro    675680685    TAA2064    (2) INFORMATION FOR SEQ ID NO:33:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 687 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    AspAsnGlnAsnIleProIleThrAspValAspValArgSerHisGlu    151015    AsnAsnLeuAlaHisArgThrArgGluIleAspArgGluArgLeuIle    202530    ValArgArgGlyGlnProPheSerIleSerLeuGlnCysCysAspSer    354045    LeuThrArgAsnHisHisLeuGluLeuSerLeuHisLeuGlyLysLys    505560    AspGluValValIleLysValHisAsnGluProGluAlaGlyGlyLys    65707580    TrpTrpPheAsnHisGlnLysValGlnAspGluIleLeuLeuThrLeu    859095    HisSerProAlaAspAlaIleIleGlyGluTyrHisLeuThrValLeu    100105110    IleLysSerProAspGlyHisPheValLysLysThrLysAsnIleGly    115120125    PheHisLeuLeuPheAsnProTrpCysLysAspAspAlaValTyrLeu    130135140    ProAspGluArgMetLeuAspGluTyrValMetAsnGluGluGlyIle    145150155160    IleTyrArgGlyThrSerAsnHisIleSerSerIleProTrpAsnTyr    165170175    GlyGlnPheGluAspTyrValMetAspIleCysPheGlnValLeuAsp    180185190    AsnSerLysGluAlaLeuLysAsnSerLysMetAspIleGluLysArg    195200205    SerAspProValTyrValSerArgMetIleThrAlaMetValAsnSer    210215220    AsnGlyAspArgGlyValLeuThrGlyGlnTrpHisGluProTyrThr    225230235240    GlyGlyPheSerProLeuArgTrpThrGlySerValProIleLeuArg    245250255    LysTrpSerLysAlaGluValArgAlaValLysTyrGlyGlnCysTrp    260265270    ValPheAlaAlaValAlaCysThrValLeuArgCysLeuGlyIlePro    275280285    ThrArgAsnIleThrAsnPheAsnSerAlaHisAspValAspGlyAsn    290295300    LeuSerValAspIleValLeuAsnLysGluMetGluSerValGlyLys    305310315320    LysAspSerSerTrpAsnPheHisCysTrpIleGluSerTrpMetArg    325330335    ArgAspAspLeuSerLysGlyAsnAspGlyTrpGlnValLeuAspPro    340345350    ThrProGlnGluLeuSerAspGlyGluTyrCysCysGlyProCysPro    355360365    ValThrAlaIleLysGluGlyAsnLeuSerValLysTyrAspAlaPro    370375380    PheIlePheAlaGluValAsnAlaAspIleIleTyrTrpMetAlaGly    385390395400    ProGlyGlyGluArgLysLysIleAspValAspGlnSerGlyValGly    405410415    LysAsnIleSerThrLysSerLeuTyrGlyAspTyrArgGluAspVal    420425430    ThrLeuHisTyrLysTyrProGluGlySerLysLysGluArgGluVal    435440445    TyrGlnLysAlaGlyHisArgIleLysGluGlnIleCysGluAsnLys    450455460    GlyProGlnGlnLeuGlnLeuSerValLysHisGlyLysProValPhe    465470475480    GlyThrAspPheAspValIleValGluValLysAsnGluGlyGlnLys    485490495    AspThrSerProGlnLeuLeuIleValValMetAlaValThrTyrAsn    500505510    SerIleAsnGlnGlyGluCysGlnArgLysAlaThrIleValThrVal    515520525    ProAlaArgLysThrHisLysGluValLeuArgLeuArgTyrAspAsp    530535540    TyrValLysCysValSerGluHisHisLeuIleArgValLysAlaLeu    545550555560    MetGluValProGlyAspAsnLysProValMetSerValAlaAsnIle    565570575    ProLeuSerMetProGluLeuLeuValGluValProGlySerIleIle    580585590    ValGlnGluLysValThrAlaPheIleSerPheThrAsnProLeuThr    595600605    ValProLeuLysArgGlyMetPheThrValGluGlySerGlyLeuLeu    610615620    SerAlaSerGluIleTyrValLysGlyAspIleAlaProGlyGlnLys    625630635640    ValSerValLysIleThrPheThrProMetArgValGlyValArgLys    645650655    LeuLeuValAspPheAspSerAspArgLeuLysAspValLysGlyVal    660665670    ThrThrValValValArgLysLysSerCysPheIleArgCysPro    675680685    (2) INFORMATION FOR SEQ ID NO:34:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    GluIleAspArg    (2) INFORMATION FOR SEQ ID NO:35:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    ValTyrLeuProAspGlu    15    (2) INFORMATION FOR SEQ ID NO:36:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    ValMetAspIleCysPhe    15    (2) INFORMATION FOR SEQ ID NO:37:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:    AspGlyAsnLeuSerXaaAsp    15    (2) INFORMATION FOR SEQ ID NO:38:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:    AspSerXaaTrpAsnPheHisCysTrpXaaGluSer    1510    (2) INFORMATION FOR SEQ ID NO:39:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:    GlyTrpGlnValLeuAspPro    15    (2) INFORMATION FOR SEQ ID NO:40:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:    LysGluArgGluValTyrXaaLysAlaGly    1510    (2) INFORMATION FOR SEQ ID NO:41:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:    ProValPheGlyThrAspPheAspValIle    1510    (2) INFORMATION FOR SEQ ID NO:42:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:    AlaValThrTyrAsnSer    15    (2) INFORMATION FOR SEQ ID NO:43:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:    ProAlaXaaLysXaaHis    15    (2) INFORMATION FOR SEQ ID NO:44:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:    ValSerGluHis    1    (2) INFORMATION FOR SEQ ID NO:45:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 9 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:    LeuValAspPheAspSerAspArgLeu    15    (2) INFORMATION FOR SEQ ID NO:46:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Theragra chalcogramma    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:    XaaAlaGlyGlySerGlyAsp    15    (2) INFORMATION FOR SEQ ID NO:47:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:    TrpTrpLeuHisGlnGlnSer    15    (2) INFORMATION FOR SEQ ID NO:48:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:    MetTyrLeuLeuPheAsnPro    15    (2) INFORMATION FOR SEQ ID NO:49:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 8 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:    TrpGlnGluProTyrThrGlyGly    15    (2) INFORMATION FOR SEQ ID NO:50:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 13 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:    PheAspValProPheValPheAlaGluValAsnAlaAsp    1510    (2) INFORMATION FOR SEQ ID NO:51:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:    SerXaaTyrSerAsnGlu    15    (2) INFORMATION FOR SEQ ID NO:52:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:    HisHisLeuGluLeuValLeuXaaLeuGly    1510    (2) INFORMATION FOR SEQ ID NO:53:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:    XaaXaaPheAsnGlnGlnGlyAlaGlnAspGluIleLeuLeuThrLeu    151015    His    (2) INFORMATION FOR SEQ ID NO:54:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 9 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:    IleSerPheHisMetLeuPheAsnPro    15    (2) INFORMATION FOR SEQ ID NO:55:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:    LeuGlnGluTyrValMetAsnGluAspGlyValIleTyrMetGlyThr    151015    (2) INFORMATION FOR SEQ ID NO:56:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:    AsnSerGluMetAspIleGluHisArgSerAspProValTyrValGly    151015    ArgThr    (2) INFORMATION FOR SEQ ID NO:57:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:    TyrAspAlaProPheValPheAlaGluValAsnAlaAspThrIleTyr    151015    (2) INFORMATION FOR SEQ ID NO:58:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:    SerValTyrGlyAsnHisArgGluAspValThrLeuHisTyr    1510    (2) INFORMATION FOR SEQ ID NO:59:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:    AlaGlyArgArgValThrGluProSerAsnGluIleAlaIleGlnGly    151015    ArgLeu    (2) INFORMATION FOR SEQ ID NO:60:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 15 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:    XaaAlaGlnProValPheGlyThrAspPheAspValIleValGlu    151015    (2) INFORMATION FOR SEQ ID NO:61:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:    AsnGluGlyGlyArgAspAlaHisAlaGlnLeuThrXaaLeuAlaXaa    151015    Ala    (2) INFORMATION FOR SEQ ID NO:62:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 9 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:    ThrIleSerValThrValProAlaHis    15    (2) INFORMATION FOR SEQ ID NO:63:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 9 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:    AlaValValXaaGluProLeuThrAla    15    (2) INFORMATION FOR SEQ ID NO:64:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:    GlyGlyValPheThrLeuGluGlyAlaGlyLeuLeuSerAlaThrGln    151015    IleHis    (2) INFORMATION FOR SEQ ID NO:65:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:    LeuSerPheSerProMetArgThrGlyValArg    1510    (2) INFORMATION FOR SEQ ID NO:66:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 10 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:    LeuLeuValAspPheAspSerAspArgLeu    1510    (2) INFORMATION FOR SEQ ID NO:67:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 8 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:    GlyValThrThrValValValHis    15    (2) INFORMATION FOR SEQ ID NO:68:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:    TyrArgSerLeuIleThrGlyLeuHisThrAsp    1510    (2) INFORMATION FOR SEQ ID NO:69:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2148 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA to mRNA    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Paralichthys olivaceus    (F) TISSUE TYPE: liver    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 26..2092    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:    GAGAAGACGAGGAAAAAGGTCTGCGATGGACAATCAGAACATTCCGATCACT52    MetAspAsnGlnAsnIleProIleThr    15    GATGTGGATGTGAGAAGTCATGAAAACAACTTGGCTCACCGCACCAGG100    AspValAspValArgSerHisGluAsnAsnLeuAlaHisArgThrArg    10152025    GAGATTGATCGGGAGCGCTTGATCGTCCGCAGGGGTCAACCCTTCTCC148    GluIleAspArgGluArgLeuIleValArgArgGlyGlnProPheSer    303540    ATATCTCTGCAGTGCTGCGACTCGCTGACCCGGAATCACCATCTGGAA196    IleSerLeuGlnCysCysAspSerLeuThrArgAsnHisHisLeuGlu    455055    CTGTCCCTGCACCTCGGTAAGAAAGATGAGGTGGTGATTAAGGTGCAC244    LeuSerLeuHisLeuGlyLysLysAspGluValValIleLysValHis    606570    AATGAGCCTGAGGCTGGAGGCAAGTGGTGGTTTAACCATCAGAAAGTG292    AsnGluProGluAlaGlyGlyLysTrpTrpPheAsnHisGlnLysVal    758085    CAGGATGAAATTCTGCTGACTCTACACAGTCCAGCGGACGCCATAATT340    GlnAspGluIleLeuLeuThrLeuHisSerProAlaAspAlaIleIle    9095100105    GGCGAGTACCACCTGACTGTGTTGATCAAGTCACCGGATGGACACTTT388    GlyGluTyrHisLeuThrValLeuIleLysSerProAspGlyHisPhe    110115120    GTGAAGAAGACTAAGAACATTGGATTCCACCTGCTCTTTAACCCCTGG436    ValLysLysThrLysAsnIleGlyPheHisLeuLeuPheAsnProTrp    125130135    TGCAAAGATGATGCTGTGTACCTCCCTGATGAAAGGATGCTCGACGAG484    CysLysAspAspAlaValTyrLeuProAspGluArgMetLeuAspGlu    140145150    TATGTTATGAATGAGGAGGGGATCATTTACAGGGGAACCTCGAATCAC532    TyrValMetAsnGluGluGlyIleIleTyrArgGlyThrSerAsnHis    155160165    ATCAGTAGCATACCCTGGAATTACGGACAGTTTGAGGACTATGTGATG580    IleSerSerIleProTrpAsnTyrGlyGlnPheGluAspTyrValMet    170175180185    GACATCTGTTTTCAAGTTCTGGACAACTCCAAGGAAGCCCTGAAGAAT628    AspIleCysPheGlnValLeuAspAsnSerLysGluAlaLeuLysAsn    190195200    TCAAAGATGGACATTGAGAAGAGATCTGACCCTGTCTATGTCAGCAGG676    SerLysMetAspIleGluLysArgSerAspProValTyrValSerArg    205210215    ATGATCACTGCGATGGTGAACTCTAACGGTGACAGGGGTGTGCTGACT724    MetIleThrAlaMetValAsnSerAsnGlyAspArgGlyValLeuThr    220225230    GGTCAGTGGCACGAGCCATACACTGGCGGGTTCTCACCACTTCGATGG772    GlyGlnTrpHisGluProTyrThrGlyGlyPheSerProLeuArgTrp    235240245    ACCGGCAGCGTGCCCATCCTCCGGAAGTGGAGCAAGGCCGAGGTCAGG820    ThrGlySerValProIleLeuArgLysTrpSerLysAlaGluValArg    250255260265    GCGGTCAAATATGGCCAGTGCTGGGTGTTTGCTGCTGTCGCCTGCACA868    AlaValLysTyrGlyGlnCysTrpValPheAlaAlaValAlaCysThr    270275280    GTGCTGCGTTGTCTGGGAATCCCAACACGCAACATCACTAACTTCAAT916    ValLeuArgCysLeuGlyIleProThrArgAsnIleThrAsnPheAsn    285290295    TCAGCACATGATGTCGATGGAAACCTCTCCGTCGACATCGTGTTGAAC964    SerAlaHisAspValAspGlyAsnLeuSerValAspIleValLeuAsn    300305310    AAAGAAATGGAGAGCGTTGGCAAGAAGGACAGTAGCTGGAACTTCCAC1012    LysGluMetGluSerValGlyLysLysAspSerSerTrpAsnPheHis    315320325    TGTTGGATCGAGTCCTGGATGAGGAGAGACGACCTCTCTAAAGGAAAT1060    CysTrpIleGluSerTrpMetArgArgAspAspLeuSerLysGlyAsn    330335340345    GACGGCTGGCAGGTTTTGGACCCCACCCCTCAAGAACTGAGTGATGGT1108    AspGlyTrpGlnValLeuAspProThrProGlnGluLeuSerAspGly    350355360    GAGTATTGCTGCGGCCCGTGTCCAGTCACCGCCATCAAGGAGGGAAAT1156    GluTyrCysCysGlyProCysProValThrAlaIleLysGluGlyAsn    365370375    CTGAGTGTGAAGTACGACGCTCCGTTTATCTTCGCTGAGGTGAACGCT1204    LeuSerValLysTyrAspAlaProPheIlePheAlaGluValAsnAla    380385390    GACATCATCTACTGGATGGCTGGACCAGGAGGCGAACGGAAGAAGATC1252    AspIleIleTyrTrpMetAlaGlyProGlyGlyGluArgLysLysIle    395400405    GATGTGGACCAGAGTGGTGTGGGGAAGAACATCAGCACCAAAAGTCTT1300    AspValAspGlnSerGlyValGlyLysAsnIleSerThrLysSerLeu    410415420425    TATGGCGACTACAGGGAGGATGTCACTCTGCACTACAAATACCCCGAA1348    TyrGlyAspTyrArgGluAspValThrLeuHisTyrLysTyrProGlu    430435440    GGCTCCAAGAAGGAGAGAGAGGTGTACCAGAAGGCCGGACACCGAATC1396    GlySerLysLysGluArgGluValTyrGlnLysAlaGlyHisArgIle    445450455    AAAGAGCAGATCTGTGAAAACAAAGGTCCACAACAACTGCAGCTGTCA1444    LysGluGlnIleCysGluAsnLysGlyProGlnGlnLeuGlnLeuSer    460465470    GTCAAGCACGGGAAACCTGTATTTGGCACTGACTTCGATGTGATAGTT1492    ValLysHisGlyLysProValPheGlyThrAspPheAspValIleVal    475480485    GAGGTGAAGAATGAAGGACAGAAAGACACCAGTCCACAGCTGCTGATT1540    GluValLysAsnGluGlyGlnLysAspThrSerProGlnLeuLeuIle    490495500505    GTGGTCATGGCCGTGACCTACAATTCCATCAATCAAGGGGAGTGTCAG1588    ValValMetAlaValThrTyrAsnSerIleAsnGlnGlyGluCysGln    510515520    AGGAAGGCGACCATAGTGACCGTGCCGGCTCGCAAAACCCACAAGGAA1636    ArgLysAlaThrIleValThrValProAlaArgLysThrHisLysGlu    525530535    GTGCTGCGTCTGCGCTACGACGACTATGTCAAATGTGTCTCTGAGCAC1684    ValLeuArgLeuArgTyrAspAspTyrValLysCysValSerGluHis    540545550    CATCTGATCAGGGTGAAAGCGCTCATGGAGGTTCCAGGGGACAACAAA1732    HisLeuIleArgValLysAlaLeuMetGluValProGlyAspAsnLys    555560565    CCCGTCATGAGTGTGGCCAACATTCCACTGAGCATGCCTGAGCTCCTG1780    ProValMetSerValAlaAsnIleProLeuSerMetProGluLeuLeu    570575580585    GTAGAGGTACCTGGGAGCATCATTGTTCAGGAGAAGGTGACAGCCTTC1828    ValGluValProGlySerIleIleValGlnGluLysValThrAlaPhe    590595600    ATCTCCTTCACAAATCCTCTAACTGTCCCACTGAAGCGTGGCATGTTC1876    IleSerPheThrAsnProLeuThrValProLeuLysArgGlyMetPhe    605610615    ACCGTTGAGGGGTCCGGACTACTGTCTGCCTCTGAGATCTATGTGAAA1924    ThrValGluGlySerGlyLeuLeuSerAlaSerGluIleTyrValLys    620625630    GGGGACATTGCTCCAGGCCAGAAGGTTTCTGTCAAGATCACCTTCACG1972    GlyAspIleAlaProGlyGlnLysValSerValLysIleThrPheThr    635640645    CCCATGAGGGTCGGGGTGAGGAAGCTCCTGGTGGACTTTGACTCTGAC2020    ProMetArgValGlyValArgLysLeuLeuValAspPheAspSerAsp    650655660665    AGGCTGAAGGATGTGAAAGGAGTCACGACAGTGGTCGTCCGCAAGAAA2068    ArgLeuLysAspValLysGlyValThrThrValValValArgLysLys    670675680    TCCTGTTTTATTAGGTGTCCTTAAAAACAGACGGACACGTATTAAAGTGTG2119    SerCysPheIleArgCysPro    685    AGATAACCTGAGAGGTGTAACTCCCCTGT2148    (2) INFORMATION FOR SEQ ID NO:70:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 688 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:    MetAspAsnGlnAsnIleProIleThrAspValAspValArgSerHis    151015    GluAsnAsnLeuAlaHisArgThrArgGluIleAspArgGluArgLeu    202530    IleValArgArgGlyGlnProPheSerIleSerLeuGlnCysCysAsp    354045    SerLeuThrArgAsnHisHisLeuGluLeuSerLeuHisLeuGlyLys    505560    LysAspGluValValIleLysValHisAsnGluProGluAlaGlyGly    65707580    LysTrpTrpPheAsnHisGlnLysValGlnAspGluIleLeuLeuThr    859095    LeuHisSerProAlaAspAlaIleIleGlyGluTyrHisLeuThrVal    100105110    LeuIleLysSerProAspGlyHisPheValLysLysThrLysAsnIle    115120125    GlyPheHisLeuLeuPheAsnProTrpCysLysAspAspAlaValTyr    130135140    LeuProAspGluArgMetLeuAspGluTyrValMetAsnGluGluGly    145150155160    IleIleTyrArgGlyThrSerAsnHisIleSerSerIleProTrpAsn    165170175    TyrGlyGlnPheGluAspTyrValMetAspIleCysPheGlnValLeu    180185190    AspAsnSerLysGluAlaLeuLysAsnSerLysMetAspIleGluLys    195200205    ArgSerAspProValTyrValSerArgMetIleThrAlaMetValAsn    210215220    SerAsnGlyAspArgGlyValLeuThrGlyGlnTrpHisGluProTyr    225230235240    ThrGlyGlyPheSerProLeuArgTrpThrGlySerValProIleLeu    245250255    ArgLysTrpSerLysAlaGluValArgAlaValLysTyrGlyGlnCys    260265270    TrpValPheAlaAlaValAlaCysThrValLeuArgCysLeuGlyIle    275280285    ProThrArgAsnIleThrAsnPheAsnSerAlaHisAspValAspGly    290295300    AsnLeuSerValAspIleValLeuAsnLysGluMetGluSerValGly    305310315320    LysLysAspSerSerTrpAsnPheHisCysTrpIleGluSerTrpMet    325330335    ArgArgAspAspLeuSerLysGlyAsnAspGlyTrpGlnValLeuAsp    340345350    ProThrProGlnGluLeuSerAspGlyGluTyrCysCysGlyProCys    355360365    ProValThrAlaIleLysGluGlyAsnLeuSerValLysTyrAspAla    370375380    ProPheIlePheAlaGluValAsnAlaAspIleIleTyrTrpMetAla    385390395400    GlyProGlyGlyGluArgLysLysIleAspValAspGlnSerGlyVal    405410415    GlyLysAsnIleSerThrLysSerLeuTyrGlyAspTyrArgGluAsp    420425430    ValThrLeuHisTyrLysTyrProGluGlySerLysLysGluArgGlu    435440445    ValTyrGlnLysAlaGlyHisArgIleLysGluGlnIleCysGluAsn    450455460    LysGlyProGlnGlnLeuGlnLeuSerValLysHisGlyLysProVal    465470475480    PheGlyThrAspPheAspValIleValGluValLysAsnGluGlyGln    485490495    LysAspThrSerProGlnLeuLeuIleValValMetAlaValThrTyr    500505510    AsnSerIleAsnGlnGlyGluCysGlnArgLysAlaThrIleValThr    515520525    ValProAlaArgLysThrHisLysGluValLeuArgLeuArgTyrAsp    530535540    AspTyrValLysCysValSerGluHisHisLeuIleArgValLysAla    545550555560    LeuMetGluValProGlyAspAsnLysProValMetSerValAlaAsn    565570575    IleProLeuSerMetProGluLeuLeuValGluValProGlySerIle    580585590    IleValGlnGluLysValThrAlaPheIleSerPheThrAsnProLeu    595600605    ThrValProLeuLysArgGlyMetPheThrValGluGlySerGlyLeu    610615620    LeuSerAlaSerGluIleTyrValLysGlyAspIleAlaProGlyGln    625630635640    LysValSerValLysIleThrPheThrProMetArgValGlyValArg    645650655    LysLeuLeuValAspPheAspSerAspArgLeuLysAspValLysGly    660665670    ValThrThrValValValArgLysLysSerCysPheIleArgCysPro    675680685    (2) INFORMATION FOR SEQ ID NO:71:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 2148 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA to mRNA    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Paralichthys olivaceus    (F) TISSUE TYPE: liver    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 26..2092    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:    GAGAAGACGAGGAAAAAGGTCTGCGATGGACAATCAGAACATTCCGATCACT52    MetAspAsnGlnAsnIleProIleThr    15    GATGTGGATGTGAGAAGTCATGAAAACAACTTGGCTCACCGCACCAGG100    AspValAspValArgSerHisGluAsnAsnLeuAlaHisArgThrArg    10152025    GAGATTGATCGGGAGCGCTTGATCGTCCGCAGGGGTCAACCCTTCTCC148    GluIleAspArgGluArgLeuIleValArgArgGlyGlnProPheSer    303540    ATATCTCTGCAGTGCTGCGACTCGCTGACCCGGAATCACCATCTGGAA196    IleSerLeuGlnCysCysAspSerLeuThrArgAsnHisHisLeuGlu    455055    CTGTCCCTGCACCTCGGTAAGAAAGATGAGGTGGTGATTAAGGTGCAC244    LeuSerLeuHisLeuGlyLysLysAspGluValValIleLysValHis    606570    AATGAGCCTGAGGCTGGAGGCAAGTGGTGGTTTAACCATCAGAAAGTG292    AsnGluProGluAlaGlyGlyLysTrpTrpPheAsnHisGlnLysVal    758085    CAGGATGAAATTCTGCTGACTCTACACAGTCCAGCGGACGCCATAATT340    GlnAspGluIleLeuLeuThrLeuHisSerProAlaAspAlaIleIle    9095100105    GGCGAGTACCACCTGACTGTGTTGATCAAGTCACCGGATGGACACTTT388    GlyGluTyrHisLeuThrValLeuIleLysSerProAspGlyHisPhe    110115120    GTGAAGAAGACTAAGAACATTGGATTCCACCTGCTCTTTAACCCCTGG436    ValLysLysThrLysAsnIleGlyPheHisLeuLeuPheAsnProTrp    125130135    TGCAAAGATGATGCTGTGTACCTCCCTGATGAAAGGATGCTCGACGAG484    CysLysAspAspAlaValTyrLeuProAspGluArgMetLeuAspGlu    140145150    TATGTTATGAATGAGGAGGGGATCATTTACAGGGGAACCTCGAATCAC532    TyrValMetAsnGluGluGlyIleIleTyrArgGlyThrSerAsnHis    155160165    ATCAGTAGCATACCCTGGAATTACGGACAGTTTGAGGACTATGTGATG580    IleSerSerIleProTrpAsnTyrGlyGlnPheGluAspTyrValMet    170175180185    GACATCTGTTTTCAAGTTCTGGACAACTCCAAGGAAGCCCTGAAGAAT628    AspIleCysPheGlnValLeuAspAsnSerLysGluAlaLeuLysAsn    190195200    TCAAAGATGGACATTGAGAAGAGATCTGACCCTGTCTATGTCAGCAGG676    SerLysMetAspIleGluLysArgSerAspProValTyrValSerArg    205210215    ATGATCACTGCGATGGTGAACTCTAACGGTGACAGGGGTGTGCTGACT724    MetIleThrAlaMetValAsnSerAsnGlyAspArgGlyValLeuThr    220225230    GGTCAGTGGCACGAGCCATACACTGGCGGGTTCTCACCACTTCGATGG772    GlyGlnTrpHisGluProTyrThrGlyGlyPheSerProLeuArgTrp    235240245    ACCGGCAGCGTGCCCATCCTCCGGAAGTGGAGCAAGGCCGAGGTCAGG820    ThrGlySerValProIleLeuArgLysTrpSerLysAlaGluValArg    250255260265    GCGGTCAAATATGGCCAGTGCTGGGTGTTTGCTGCTGTCGCCTGCACA868    AlaValLysTyrGlyGlnCysTrpValPheAlaAlaValAlaCysThr    270275280    GTGCTGCGTTGTCTGGGAATCCCAACACGCAACATCACTAACTTCAAT916    ValLeuArgCysLeuGlyIleProThrArgAsnIleThrAsnPheAsn    285290295    TCAGCACATGATGTCGATGGAAACCTCTCCGTCGACATCGTGTTGAAC964    SerAlaHisAspValAspGlyAsnLeuSerValAspIleValLeuAsn    300305310    AAAGAAATGGAGAGCGTTGGCAAGAAGGACAGTAGCTGGAACTTCCAC1012    LysGluMetGluSerValGlyLysLysAspSerSerTrpAsnPheHis    315320325    TGTTGGATCGAGTCCTGGATGAGGAGAGACGACCTCTCTAAAGGAAAT1060    CysTrpIleGluSerTrpMetArgArgAspAspLeuSerLysGlyAsn    330335340345    GACGGCTGGCAGGTTTTGGACCCCACCCCTCAAGAACTGAGTGATGGT1108    AspGlyTrpGlnValLeuAspProThrProGlnGluLeuSerAspGly    350355360    GAGTATTGCTGCGGCCCGTGTCCAGTCACCGCCATCAAGGAGGGAAAT1156    GluTyrCysCysGlyProCysProValThrAlaIleLysGluGlyAsn    365370375    CTGAGTGTGAAGTACGACGCTCCGTTTATCTTCGCTGAGGTGAACGCT1204    LeuSerValLysTyrAspAlaProPheIlePheAlaGluValAsnAla    380385390    GACATCATCTACTGGATGGCTGGACCAGGAGGCGAACGGAAGAAGATC1252    AspIleIleTyrTrpMetAlaGlyProGlyGlyGluArgLysLysIle    395400405    GATGTGGACCAGAGTGGTGTGGGGAAGAACATCAGCACCAAAAGTCTT1300    AspValAspGlnSerGlyValGlyLysAsnIleSerThrLysSerLeu    410415420425    TATGGCGACTACAGGGAGGATGTCACTCTGCACTACAAATACCCCGAA1348    TyrGlyAspTyrArgGluAspValThrLeuHisTyrLysTyrProGlu    430435440    GGCTCCAAGAAGGAGAGAGAGGTGTACCAGAAGGCCGGACACCGAATC1396    GlySerLysLysGluArgGluValTyrGlnLysAlaGlyHisArgIle    445450455    AAAGAGCAGATCTGTGAAAACAAAGGTCCACAACAACTGCAGCTGTCA1444    LysGluGlnIleCysGluAsnLysGlyProGlnGlnLeuGlnLeuSer    460465470    GTCAAGCACGGGAAACCTGTATTTGGCACTGACTTCGATGTGATAGTT1492    ValLysHisGlyLysProValPheGlyThrAspPheAspValIleVal    475480485    GAGGTGAAGAATGAAGGACAGAAAGACACCAGTCCACAGCTGCTGATT1540    GluValLysAsnGluGlyGlnLysAspThrSerProGlnLeuLeuIle    490495500505    GTGGTCATGGCCGTGACCTACAATTCCATCAATCAAGGGGAGTGTCAG1588    ValValMetAlaValThrTyrAsnSerIleAsnGlnGlyGluCysGln    510515520    AGGAAGGCGACCATAGTGACCGTGCCGGCTCGCAAAACCCACAAGGAA1636    ArgLysAlaThrIleValThrValProAlaArgLysThrHisLysGlu    525530535    GTGCTGCGTCTGCGCTACGACGACTATGTCAAATGTGTCTCTGAGCAC1684    ValLeuArgLeuArgTyrAspAspTyrValLysCysValSerGluHis    540545550    CATCTGATCAGGGTGAAAGCGCTCATGGAGGTTCCAGGGGACAACAAA1732    HisLeuIleArgValLysAlaLeuMetGluValProGlyAspAsnLys    555560565    CCCGTCATGAGTGTGGCCAACATTCCACTGAGCATGCCTGAGCTCCTG1780    ProValMetSerValAlaAsnIleProLeuSerMetProGluLeuLeu    570575580585    GTAGAGGTACCTGGGAGCATCATTGTTCAGGAGAAGGTGACAGCCTTC1828    ValGluValProGlySerIleIleValGlnGluLysValThrAlaPhe    590595600    ATCTCCTTCACAAATCCTCTAACTGTCCCACTGAAGCGTGGCATGTTC1876    IleSerPheThrAsnProLeuThrValProLeuLysArgGlyMetPhe    605610615    ACCGTGGAGGGGTCCGGACTACTGTCTGCCTCTGAGATCTATGTGAAA1924    ThrValGluGlySerGlyLeuLeuSerAlaSerGluIleTyrValLys    620625630    GGGGACATTGCTCCAGGCCAGAAGGTTTCTGTCAAGATCACCTTCACG1972    GlyAspIleAlaProGlyGlnLysValSerValLysIleThrPheThr    635640645    CCCATGAGGGTCGGGGTGAGGAAGCTCCTGGTGGACTTTGACTCTGAC2020    ProMetArgValGlyValArgLysLeuLeuValAspPheAspSerAsp    650655660665    AGGCTGAAGGATGTGAAAGGAGTCACGACAGTGGTCGTCCGCAAGAAA2068    ArgLeuLysAspValLysGlyValThrThrValValValArgLysLys    670675680    TCCTGTTTTATTAGGTGTCCTTAAAAACAGACGGACACGTATTAAAGTGTG2119    SerCysPheIleArgCysPro    685    AGATAACCTGAGAGGTGTAACTCCCCTGT2148    (2) INFORMATION FOR SEQ ID NO:72:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 688 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:    MetAspAsnGlnAsnIleProIleThrAspValAspValArgSerHis    151015    GluAsnAsnLeuAlaHisArgThrArgGluIleAspArgGluArgLeu    202530    IleValArgArgGlyGlnProPheSerIleSerLeuGlnCysCysAsp    354045    SerLeuThrArgAsnHisHisLeuGluLeuSerLeuHisLeuGlyLys    505560    LysAspGluValValIleLysValHisAsnGluProGluAlaGlyGly    65707580    LysTrpTrpPheAsnHisGlnLysValGlnAspGluIleLeuLeuThr    859095    LeuHisSerProAlaAspAlaIleIleGlyGluTyrHisLeuThrVal    100105110    LeuIleLysSerProAspGlyHisPheValLysLysThrLysAsnIle    115120125    GlyPheHisLeuLeuPheAsnProTrpCysLysAspAspAlaValTyr    130135140    LeuProAspGluArgMetLeuAspGluTyrValMetAsnGluGluGly    145150155160    IleIleTyrArgGlyThrSerAsnHisIleSerSerIleProTrpAsn    165170175    TyrGlyGlnPheGluAspTyrValMetAspIleCysPheGlnValLeu    180185190    AspAsnSerLysGluAlaLeuLysAsnSerLysMetAspIleGluLys    195200205    ArgSerAspProValTyrValSerArgMetIleThrAlaMetValAsn    210215220    SerAsnGlyAspArgGlyValLeuThrGlyGlnTrpHisGluProTyr    225230235240    ThrGlyGlyPheSerProLeuArgTrpThrGlySerValProIleLeu    245250255    ArgLysTrpSerLysAlaGluValArgAlaValLysTyrGlyGlnCys    260265270    TrpValPheAlaAlaValAlaCysThrValLeuArgCysLeuGlyIle    275280285    ProThrArgAsnIleThrAsnPheAsnSerAlaHisAspValAspGly    290295300    AsnLeuSerValAspIleValLeuAsnLysGluMetGluSerValGly    305310315320    LysLysAspSerSerTrpAsnPheHisCysTrpIleGluSerTrpMet    325330335    ArgArgAspAspLeuSerLysGlyAsnAspGlyTrpGlnValLeuAsp    340345350    ProThrProGlnGluLeuSerAspGlyGluTyrCysCysGlyProCys    355360365    ProValThrAlaIleLysGluGlyAsnLeuSerValLysTyrAspAla    370375380    ProPheIlePheAlaGluValAsnAlaAspIleIleTyrTrpMetAla    385390395400    GlyProGlyGlyGluArgLysLysIleAspValAspGlnSerGlyVal    405410415    GlyLysAsnIleSerThrLysSerLeuTyrGlyAspTyrArgGluAsp    420425430    ValThrLeuHisTyrLysTyrProGluGlySerLysLysGluArgGlu    435440445    ValTyrGlnLysAlaGlyHisArgIleLysGluGlnIleCysGluAsn    450455460    LysGlyProGlnGlnLeuGlnLeuSerValLysHisGlyLysProVal    465470475480    PheGlyThrAspPheAspValIleValGluValLysAsnGluGlyGln    485490495    LysAspThrSerProGlnLeuLeuIleValValMetAlaValThrTyr    500505510    AsnSerIleAsnGlnGlyGluCysGlnArgLysAlaThrIleValThr    515520525    ValProAlaArgLysThrHisLysGluValLeuArgLeuArgTyrAsp    530535540    AspTyrValLysCysValSerGluHisHisLeuIleArgValLysAla    545550555560    LeuMetGluValProGlyAspAsnLysProValMetSerValAlaAsn    565570575    IleProLeuSerMetProGluLeuLeuValGluValProGlySerIle    580585590    IleValGlnGluLysValThrAlaPheIleSerPheThrAsnProLeu    595600605    ThrValProLeuLysArgGlyMetPheThrValGluGlySerGlyLeu    610615620    LeuSerAlaSerGluIleTyrValLysGlyAspIleAlaProGlyGln    625630635640    LysValSerValLysIleThrPheThrProMetArgValGlyValArg    645650655    LysLeuLeuValAspPheAspSerAspArgLeuLysAspValLysGly    660665670    ValThrThrValValValArgLysLysSerCysPheIleArgCysPro    675680685

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A DNA fragment having a gene derived from fishwhich codes for a polypeptide which possesses transglutaminase activity,said gene having a sequence which codes for a polypeptide selected fromthe group consisting of SEQ ID NO:4 and SEQ ID NO:6;wherein said fish isPagrus major.
 2. A recombinant plasmid which comprises the DNA fragmentof claim 1 and a plasmid vector.
 3. The recombinant plasmid of claim 2,wherein said plasmid is an expression vector.
 4. A transformant,transformed with the recombinant plasmid of claim
 2. 5. A method for theproduction of a fish-derived polypeptide possessing transglutaminaseactivity, comprising culturing the transformant of claim
 4. 6. Afish-derived polypeptide possessing transglutaminase activity, obtainedby culturing the transformant of claim
 4. 7. A DNA fragment having agene derived from Pagrus major having a sequence selected from the groupconsisting of SEQ ID NO:3 and SEQ ID NO:5 which codes for a polypeptidewhich possesses transglutaminase activity.